JP2023542230A - PRAME-specific T cell receptor and its uses - Google Patents

Abstract

The present invention relates to a T cell receptor (TCR) capable of binding to a polypeptide comprising the amino acid sequence LYVDSLFFL, or a portion thereof, or an HLA-A bound form thereof. The invention further relates to a nucleic acid molecule encoding said TCR, a vector comprising said nucleic acid molecule, and a host cell comprising said nucleic acid molecule or vector. The invention further relates to a method for obtaining said TCR, and to pharmaceutical and diagnostic compositions comprising said TCR, said nucleic acid molecule, vector and/or host cell. The invention further relates to such pharmaceutical and diagnostic compositions for use in diagnosing, detecting, preventing and/or treating cancer. Furthermore, the present invention relates to the use of said TCR, nucleic acid molecule, or said vector for producing modified lymphocytes.

Description

The present invention relates to a T cell receptor (TCR) capable of binding to a polypeptide, or a portion thereof, comprising the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), or an HLA-A bound form thereof. The invention further relates to a nucleic acid molecule encoding said TCR, a vector comprising said nucleic acid molecule, and a host cell comprising said nucleic acid molecule or vector. The invention further relates to a method for obtaining said TCR, and to pharmaceutical and diagnostic compositions comprising said TCR, said nucleic acid molecule, vector and/or host cell. The invention further relates to such pharmaceutical and diagnostic compositions for use in diagnosing, detecting, preventing and/or treating cancer. Furthermore, the present invention relates to the use of said TCR, nucleic acid molecule, or said vector for producing modified lymphocytes.

T lymphocytes (or T cells), which form part of the cell-mediated immune system, play a major role in eradicating pathogens. T cells develop in the thymus and express T cell receptor molecules on their surface, which allow them to recognize peptides presented on major histocompatibility complex (MHC) molecules expressed on nucleated cells. enable (antigen presentation). Antigens of pathogens, i.e., foreign antigens presented by MHC molecules, are thought to elicit strong T-cell responses, whereas self-antigens are thought to elicit strong T-cell responses, whereas self-antigens are highly sensitive to self-antigen-specific T-cells in the thymus during the development of such T-cells. Due to negative selection, it does not normally lead to a T cell response. Thus, the immune system can distinguish between nucleated cells presenting foreign or self antigens and can specifically target infected cells and eradicate them through potent cytokine release and cytotoxic mechanisms of T cells. .

The power of the immune system is recognized as a promising tool for future cancer therapy. In the last decade, research has begun to exploit the unique properties of T cells by using adoptive cell transfer (ACT), which involves the administration of ex vivo expanded donor-derived lymphocytes. ACT is an attractive concept for the treatment of cancer because it does not require the immune competence of the patient, and the specificity of the transferred lymphocytes is that they are typically nonmutated, unable to elicit autologous T cell responses effectively. , and therefore can be targeted against tumor antigens with low immunogenicity. Although ACT has been shown to be a promising treatment for various types of cancer, its widespread application as a clinical treatment can not only be difficult and time-consuming, but also require the use of high-avidity T cells. It is hampered by the need for custom isolation and characterization of tumor-specific T cells from each patient, a step that is often impossible to produce (Xue et al., Clin Exp Immunol. 2005 February; 139(2) : 167-172; Schmitt et al., Hum Gene Ther. 2009 November; 20(11): 1240-1248).

Gene transfer of tumor antigen-specific T cell receptors (TCR) into primary T cells allows for the rapid generation of tumor-reactive T lymphocytes with defined antigen specificity, even in immunodeficient patients. may overcome some of the current limitations of ACT. However, the identification of suitable T cell clones with TCRs that specifically recognize tumor antigens and exhibit the desired antitumor effects in vivo remains a subject of ongoing research. Given that approximately 14.1 million new cases of cancer occurred globally in 2012, and that cancer is currently responsible for approximately 14.6% of all human deaths worldwide, this is a novel and efficient method. Treatment options are urgently needed. It is an object of the present invention to meet the needs specified above.

PRAME is a tumor-associated antigen expressed in a wide variety of tumors, preferably melanoma. Furthermore, PRAME has been used as an independent biomarker of metastasis such as uveal melanoma (Fiedl et al., Clin Cancer Res 2016 March; 22(5): 1234-1242) and as a prognostic marker of DLBCL (Mitsuhashi et al. ., Hematology 2014, 1/2014). It is not expressed in normal tissues except the testis. This expression pattern is similar to that of other cancer testis (CT) antigens such as MAGE, BAGE, and GAGE. However, unlike these other CT antigens, this gene is also expressed in acute leukemia. The encoded protein acts as a retinoic acid receptor repressor and may confer a growth advantage to cancer cells through this function. Alternative splicing results in multiple transcript variants. PRAME overexpression in triple-negative breast cancer has also been found to promote cancer cell motility through the induction of epithelial-to-mesenchymal transition (Al-Khadairi et al., Journal of Translational Medicine 2019; 17: 9 ). Deletion of PRAME has been reported in chronic lymphocytic leukemia; however, this is not functionally relevant, as the gene is not expressed in B cells, and the deletion does not affect physiological immunoglobulin light chains. This is the result of reconstruction.

Given that approximately 14.1 million new cases of cancer occurred globally in 2012, and that cancer is currently responsible for approximately 14.6% of all human deaths worldwide, this is a novel and efficient method. Treatment options are urgently needed.

The technical problem underlying the invention was therefore to meet the objectives specified above. The technical problem was solved by the means and methods described herein, illustrated in the examples and defined in the claims.

The present invention relates to a polypeptide comprising or consisting of an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), in which not more than 4 amino acids have been substituted, or to a part of said polypeptide, or to said polypeptide or a T cell receptor (TCR) capable of binding to each HLA-A binding form of a portion thereof;
TCR is
(A)
(Aa) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to SEQ ID NO: 12, or preferably of a TCR alpha chain comprising or consisting of an amino acid sequence that is 100% similar or identical (preferably identical) and/or (Ab) at least 80%, 85%, 90% to SEQ ID NO: 14; comprises or consists of an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% similar or identical, or preferably 100% similar or identical (preferably identical) CDR3 of the TCR beta chain, or (B)
(Ba) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to SEQ ID NO: 40, or preferably of a TCR alpha chain comprising or consisting of an amino acid sequence that is 100% similar or identical (preferably identical) and/or (Bb) at least 80%, 85%, 90% to SEQ ID NO: 42; comprises or consists of an amino acid sequence that is 95%, 96%, 97%, 98%, or 99% similar or identical, or preferably 100% similar or identical (preferably identical) CDR3 of TCR beta chain
Regarding TCR, including.

As surprisingly found in the context of the present invention, a portion of the PRAME peptide, i.e. according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2; PRAME _301-309 ), in which not more than 4 amino acids are substituted. Polypeptides comprising or consisting of amino acid sequences are presented by cells via human leukocyte antigen class A (HLA-A) and are effectively recognized by the TCRs described and provided herein. Binding of cells comprising a TCR of the invention to said polypeptide results in significant IFN-gamma (IFN-γ) secretion by T cells transduced with a TCR of the invention and effective activation of such polypeptide-bearing cells. lead to death or injury. As further described and specified herein, a polypeptide comprising or consisting of an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2; PRAME _301-309 ) in which no more than four amino acids are substituted: Also referred to herein as "PRAME _LL -peptide".

The term "T cell receptor" or "TCR" as used herein includes the native TCR, as well as TCR variants, fragments, and constructs, in all grammatical forms. Thus, the term includes heterodimers and multimers comprising TCR alpha and beta chains, and single chain constructs, optionally comprising additional domains and/or moieties.

According to the invention, in its natural form, the TCR is present as a complex of several proteins on the surface of T cells. The T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and is composed of two (separate) protein chains called the alpha (α-) and beta (β-) chains. be done. Each chain of the TCR consists of one N-terminal immunoglobulin-like (Ig)-variable (V) domain/region, one Ig-constant-like (C) domain/region, and a transmembrane/region that anchors the chain within the plasma membrane. Possesses a region spanning the cell membrane and a short cytoplasmic tail at the C-terminus.

According to the invention, antigen specificity is conferred by the variable regions of the alpha and beta chains. Both the TCR alpha and beta chain variable domains contain three hypervariable or complementarity determining regions (CDR1 alpha/beta, CDR2 alpha/beta, and CDR3 alpha/beta) surrounded by framework (FR) regions. . CDR3 is the main determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), whereas CDR1 and CDR2 primarily interact with MHC molecules presenting antigenic peptides. do.

Natural TCRs recognize antigenic peptides bound (“presented on/displayed”) to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Antigenic peptides presented on MHC molecules are also referred to herein as "peptide:MHC complexes" or "peptide:HLA(-A) complexes." There are two different classes of MHC molecules that present peptides from different cellular compartments: MHC I and MHC II. MHC class I molecules are expressed on the surface of all nucleated cells throughout the human body and present peptides or protein fragments from intracellular compartments to cytotoxic T cells. In humans, MHC is also called human leukocyte antigen (HLA). There are three major types of MHC class I: HLA-A, HLA-B, and HLA-C. When the TCR binds to its specific peptide:MHC (eg, peptide:HLA-A) complex, the T cell is activated and exerts biological effector functions.

In one embodiment of the invention, the TCRs described and provided according to the invention have their antigenic target, i.e., according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), in which not more than 4 amino acids are substituted. specifically binds to a polypeptide comprising or consisting of an amino acid sequence (PRAME _LL -peptide), or to a portion of said polypeptide, or to the respective HLA-A binding form of said polypeptide or portion thereof. As used herein, the term "specifically binds" generally means that a TCR more readily binds to its antigen binding site than to a random, unrelated, non-target antigen. Indicates a connection via. Specific interaction of an antigen interaction site with its specific antigen may also result in simple binding of said site to the antigen. Furthermore, the specific interaction of an antigen interaction site with its specific antigen may alternatively result in the initiation of a signal, eg due to induction of conformational changes in the antigen, oligomerization of the antigen, etc. Typically, in this ^context and according to the invention, "specific binding" ^as used herein means a half-maximal IFN-γ secretion (EC ₅₀ ) refers to the functional affinity determined by Preferably, in the context of the present invention, binding is considered specific if the binding affinity is about 10 ^-11 to 10 ^-8 M (EC ₅₀ ), preferably about 10 ^-11 to 10 ^-9 M.

As indicated herein, the TCRs described and provided in the context of the present invention are presented on cells, particularly via HLA-A molecules (i.e. in their respective HLA-A bound forms). PRAME _LL - peptide or portion thereof as described and designated herein. The antigenic peptide may be complexed with HLA-A molecules, which may be presented on the surface of antigen-presenting cells such as dendritic cells or tumor cells, or which may be immobilized on beads or plates, for example by coating. , it is said to exist in its "HLA-A bound form". In the context of the present invention, such HLA-A molecules may be of any (sub)allelic type, in particular HLA-A molecules encoded by the alleles HLA-A ^* 24 or HLA-A ^* 02. including. As such, the TCRs described and provided herein are capable of binding via HLA-A ^* 24 or HLA-A ^* 02 molecules, i.e. in their respective HLA-A ^* 24 or HLA-A ^* 02 bound forms. Specifically binds to the PRAME _LL -peptide described and designated herein or a portion thereof when displayed on a cell. In specific embodiments, HLA-A ^* 24 is an HLA-A ^* 24:02 encoding molecule and/or HLA-A ^* 02 is an HLA-A ^* 02:17 encoding molecule. As such, the TCRs described and provided herein are capable of transducing HLA-A ^* 24:02 or HLA-A ^* 02:17 via their respective HLA-A ^* 24:02 or HLA-A ^* 02:17 specifically binds to the PRAME _LL -peptide or portion thereof, as described and designated herein, when presented on a cell. In a preferred specific embodiment, the TCRs described and provided herein are activated on cells via HLA-A ^* 24:02 molecules, i.e. in their respective HLA-A ^* 24:02 bound forms. specifically binds to the PRAME _LL -peptide or portion thereof as described and designated herein when presented in

According to the present invention, the term "similar" as used herein in the context of amino acid sequences means that a given amino acid sequence has the same amino acids or Meant to include only conservative or highly conservative substitutions. As used herein, "conservative" substitutions refer to the substitutions listed as "Exemplary Substitutions" in Table I below. As used herein, "highly conservative" substitutions refer to the substitutions set forth in Table I below under the heading "Preferred Substitutions."

As used herein, the term "amino acid" or "amino acid residue" typically refers to alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); Cysteine (Cys or C); Glutamine (Gln or Q); Glutamic acid (Glu or E); Glycine (Gly or G); Histidine (His or H); Isoleucine (He or I); Leucine (Leu or L ); Lysine (Lys or K); Methionine (Met or M); Phenylalanine (Phe or F); Proline (Pro or P); Serine (Ser or S); Threonine (Thr or T); Tryptophan (Trp or W) ; tyrosine (Tyr or Y); and valine (Val or V), but not modified, synthetic or rare amino acids can be used as desired. Generally, amino acids have nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); negatively charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Asp, Glu); chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr). obtain.

The term "position" as used in accordance with the present invention refers to the position of an amino acid within the amino acid sequence depicted herein. The term "corresponding" in this context also includes that the position is not determined solely by the number of preceding nucleotides/amino acids.

The level of identity between two or more sequences (eg, nucleic acid or amino acid sequences) can be readily determined by methods known in the art, such as by BLAST analysis. Generally, in the context of the present invention, "identity" is used when two sequences (e.g., polynucleotide sequences or amino acid sequences) to be compared, e.g., by sequence comparison, differ in terms of identity. The term can refer to a shorter sequence and that portion of a longer sequence that matches said shorter sequence. Therefore, if the sequences being compared do not have the same length, the degree of identity is preferably the percentage of nucleotide residues in the shorter sequence that are identical to nucleotide residues in the longer sequence, or can refer to any percentage of nucleotides in a longer sequence that are identical to a nucleotide sequence in a longer sequence. In this context, one skilled in the art is readily in a position to determine that portion of the longer sequence that matches the shorter sequence. Furthermore, as used herein, the level of identity of nucleic acid or amino acid sequences may refer to the entire length of the respective sequences, preferably pairwise, with each gap being counted as one mismatch. be assessed. These definitions for sequence comparisons (eg, establishing "identity" values) should apply to all sequences described and disclosed herein.

Additionally, the term "identity" as used herein means that there is functional and/or structural equivalence between corresponding sequences. Nucleic acid/amino acid sequences having a given level of identity with particular nucleic acid/amino acid sequences described herein may be derivatives/variants of these sequences, preferably having the same biological function. They may be either naturally occurring variations, such as sequences derived from other subspecies, species, etc., or mutations, said mutations may be formed naturally or by deliberate mutagenesis. It may be produced. Additionally, the variation may be a synthetically produced sequence. A variant can be a naturally occurring variant or a synthetically produced variant or a variant produced by recombinant DNA techniques.

As used herein, "deviations" from a sequence (eg, an amino acid or nucleic acid sequence) can include, for example, deletions, substitutions, additions, insertions, and/or recombinations. The term "addition" refers to the addition of a nucleic acid residue/amino acid to the end or beginning of a given sequence, whereas "insertion" refers to the insertion of a nucleic acid residue/amino acid within a given sequence. refers to something. The term "deletion" refers to the deletion or removal of a nucleic acid or amino acid residue in a given sequence. The term "substitution" refers to the replacement of nucleic acid/amino acid residues in a given sequence. Again, these definitions as used herein apply mutatis mutandis to all sequences provided and described herein, unless otherwise specified.

In one embodiment of the invention, the TCR of the invention binds (specifically) comprises or consists of an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), in which not more than 4 amino acids are substituted. In the polypeptide that does, positions 2Y and 8F are not substituted. In another embodiment of the invention, position 6L is the only conservative or preferably highly conservative substitution, or even more preferably is not substituted. In a specific embodiment of the invention, said polypeptide (PRAME _LL -peptide) does not have substitutions at positions 2Y, 6L and 8F (relative to SEQ ID NO: 2). In a more specific embodiment of the invention, said polypeptide (PRAME _LL -peptide) does not have substitutions at positions 2Y, 5S, 6L, 7F and 8F (relative to SEQ ID NO: 2).

The term "polypeptide" is used herein equivalently to the term "protein" or "peptide" unless specifically indicated otherwise. Proteins (including fragments thereof, preferably biologically active fragments, and peptides usually having less than 30 amino acids) are proteins linked to each other via covalent peptide bonds (resulting in chains of amino acids). or contains multiple amino acids. The term "polypeptide" as used herein describes a group of molecules that typically contain more than 15 amino acids. Polypeptides may further form multimers, such as dimers, trimers, and higher order oligomers, ie, consist of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher order structures of such multimers are therefore referred to as homo- or heterodimers, homo- or heterotrimers, and the like. An example for a heteromultimer is an antibody molecule which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides/proteins, where the modification results from post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well known in the art.

As used herein, in the context of polypeptides, the term "portion" (of a given polypeptide) includes deletions of the N-terminal and/or C-terminal portions of such polypeptide. good, refers to a contiguous portion of such a polypeptide. Preferably, as used herein, a "portion" comprises at least 5, more preferably 6 or 7, most preferably at least 8 contiguous amino acids of said polypeptide. According to the invention, such a "moiety" is preferably a "functional moiety", i.e. it is still recognized by the TCR as described and provided herein through (specific) binding. and preferably induces IFN-γ secretion by cells containing said TCR.

In one embodiment of the invention, to the PRAME _LL -peptide or portion thereof as described and further specified herein, or in its HLA-A-bound form as described and specified herein; Binding of the TCR described and provided herein induces IFN-γ secretion by cells containing said TCR. In one embodiment, in this context and according to the invention, the level of IFN-γ secretion of such cells comprising a TCR of the invention as compared to control cells not comprising said TCR or in an unrelated as described herein and further specified as compared to a cell comprising said TCR binding to a peptide (i.e., a peptide that is not a PRAME _LL -peptide or a portion thereof as described and further specified herein). binding to the PRAME _L-L -peptide (or a portion thereof, or its HLA-A binding form as described and designated herein) at least 3-fold, preferably at least 5-fold, 10-fold , or 20 times higher. In the context of the present invention, and also as described and exemplified herein, the measurement of IFN-gamma may be performed by any suitable method known in the art, such as ELISA. By way of example, for such assays (e.g., ELISA), the concentration of PRAME _LL -peptide (and unrelated peptide as a control) can be about 10 ^-5 M, with cells containing TCR versus target (alone). or in its HLA-A bound form as described and designated herein, the ratio of PRAME _{LL -} peptide or portion thereof) may be about 1:2. Cells containing TCRs described and provided herein may be capable of producing such TCRs, either naturally or preferably by transduction, transfection, or any other suitable method of stably inserting a nucleic acid molecule into the cell. It may also receive a nucleic acid molecule encoding a TCR. Suitable cells containing said TCR are known in the art and are further described and provided herein as "host cells." Suitable target cells presenting the PRAME _LL -peptide or portion thereof as described and specified herein are preferably presented in their HLA-A bound form via an HLA-A molecule. It encodes an HLA _{-A molecule in order to be able to present said PRAME LL} -peptide or a portion thereof as described and specified. In this context and according to the invention, as described herein, specific examples for HLA-A are HLA-A ^* 24 (e.g., HLA-A ^* 24:02) and HLA-A ^* 02 (eg, HLA-A ^* 02:17).

According to the present invention, the TCRs described and provided herein (e.g., native TCRs) are preferably expressed by their antigenic targets (i.e., e.g., HLA-A ^* 24 (e.g., HLA- A ^* 24:02) or HLA-A ^* 02 (e.g. HLA-A ^* 02:17) encoding molecules, preferably presented on HLA-A ^* 24:02 encoding molecules by antigen presenting cells. PRAME _LL - peptide or a portion thereof, or its HLA-A-bound form), with high functional avidity. The term "functional avidity" refers to the ability of a TCR-expressing cell (particularly a T cell expressing a native TCR as described herein) to respond in vitro to a given concentration of a ligand; It is thought that this correlates with the in vivo effector ability of By definition, TCR-expressing cells with high functional avidity will respond to very low antigen doses in in vitro tests, whereas such cells with lower functional avidity will A higher amount of antigen is required before mounting an immune response comparable to that of TCR-expressing cells. Therefore, functional avidity can be considered a quantitative determinant of the activation threshold of TCR-expressing cells. It is determined by exposing such cells in vitro to varying amounts of the cognate antigen. TCR expressing cells with high functional avidity respond to low antigen doses.

For example, a TCR-expressing cell may have a molecular weight of about 10 ⁻⁵ to about 10 ⁻¹¹ M (i.e., about 0.05 ng/mL to about 5 ng/mL), which has a molecular weight of a PRAME peptide having an amino acid sequence according to SEQ ID NO: 2. antigen-negative HLA-A (e.g., HLA- When co-cultured with A ^* 24 (e.g., HLA-A ^* 24:02) or HLA-A ^* 02 (e.g., HLA-A ^* 02:17)) expressing target cells, it is estimated that (e.g., 200 pg/mL or more, 300 pg/mL or more, 400 pg/mL or more, 500 pg/mL or more, 600 pg/mL or more, 700 pg/mL or more) interferon gamma (IFN-gamma) If it is secreted, it will typically be considered to bind to its antigenic target with "high" functional avidity. Therefore, the TCR of the present invention is a TCR with high functional avidity that causes a half-maximal relative IFN-γ secretion (EC50 value) of less than 10 ⁻⁵ M, as measured by IFN-gamma immunoassay. Preferably, the half-maximal relative IFN-gamma secretion (EC50 value) evoked is less than 10 ⁻⁶ M as measured by IFN-gamma immunoassay (see Figure 4 and Example 4).

Cytokine release, such as IFN-gamma secretion, is performed by any means known in the art and also otherwise exemplified herein, or by, for example, HLA-A ^* 24:02 or HLA-A ^* 02. :17 donor-derived LCLs are transfected or transduced with ivtRNA to express, for example, the amino acid sequence of SEQ ID NO: 2 or an unrelated peptide, respectively, CD8 ⁺ enriched and/or expressing the TCR to be investigated. or using an in vitro assay by incubating with non-CD8 ⁺ enriched PBMCs or externally carrying either the PRAME peptide according to SEQ ID NO: 2 or an unrelated peptide and subsequently expressing the TCR to be investigated. It can be determined in an in vitro assay using T2 cells co-incubated with CD8 ⁺ enriched and/or non-CD8 ⁺ enriched PBMC.

In one embodiment of the invention, the TCR described and provided herein comprising a CDR3 according to (A) further comprises a corresponding CDR1 and/or CDR2 subregion. In one embodiment of the invention, the TCR described and provided herein comprising CDR3 according to (A) is
(Aa1) At least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to or identical to the amino acid sequence of SEQ ID NO: 4 (preferably identical) CDR1 of the TCR alpha chain comprising or consisting of the amino acid sequence and/or at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to the amino acid sequence of SEQ ID NO: 8. CDR2 of the TCR alpha chain comprising or consisting of an amino acid sequence that is or is the same (preferably the same) and/or (Ab1) at least 80%, 85%, 90%, 95% with the amino acid sequence of SEQ ID NO: 6. %, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to the CDR1 of the TCR beta chain, and/or the amino acid of SEQ ID NO: 10. A TCR comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to the sequence Beta chain CDR2
further including.

In another embodiment of the invention, the TCR described and provided herein comprising a CDR3 according to (B) further comprises a corresponding CDR1 and/or CDR2 subregion. In one embodiment of the invention, the TCR described and provided herein comprising CDR3 according to (B) is
(Ba1) At least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical to (preferably identical) the amino acid sequence of SEQ ID NO: 32 CDR1 of the TCR alpha chain comprising or consisting of the amino acid sequence and/or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to the amino acid sequence of SEQ ID NO: 36. CDR2 of the TCR alpha chain comprising or consisting of an amino acid sequence that is or is the same (preferably the same) and/or (Bb1) at least 80%, 85%, 90%, 95% with the amino acid sequence of SEQ ID NO: 34. %, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to the CDR1 of the TCR beta chain, and/or the amino acid of SEQ ID NO: 38. A TCR comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to the sequence Beta chain CDR2
further including.

In one embodiment of the invention, the TCR described and provided herein comprising CDR3 according to (A) is
(Aa2) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to SEQ ID NO: 16; Contains or consists of
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 47-51 of SEQ ID NO: 16 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 69-75 of SEQ ID NO: 16 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 109-123 of SEQ ID NO: 16 a TCR alpha chain variable region comprising or consisting of a sequence and/or (Ab2) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 18; comprising or consisting of an amino acid sequence that is or is the same (preferably is the same);
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 46-50 of SEQ ID NO: 18 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 68-73 of SEQ ID NO: 18 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 110-122 of SEQ ID NO: 18 TCR beta chain variable region comprising or consisting of a sequence.

In another embodiment of the invention, the TCR described and provided herein comprising CDR3 according to (B) is
(Ba2) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to SEQ ID NO: 44; Contains or consists of
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 45 to 49 of SEQ ID NO: 44 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 67-73 of SEQ ID NO: 44 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 107-121 of SEQ ID NO: 44 a TCR alpha chain variable region comprising or consisting of a sequence and/or (Bb2) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 46; comprising or consisting of an amino acid sequence that is or is the same (preferably is the same);
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 44-49 of SEQ ID NO: 46 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 67-71 of SEQ ID NO: 46 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 108-122 of SEQ ID NO: 46 TCR beta chain variable region comprising or consisting of a sequence.

In one embodiment of the invention, the TCR described and provided herein further comprises (i) a TCR alpha chain constant region, and/or (ii) a TCR beta chain constant region. In one embodiment, the TCR alpha constant region and/or the TCR beta chain constant region may be murine (murC), e.g. SEQ ID NO: 24 and SEQ ID NO: 26, respectively, minimally murine (mmC), e.g. SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or may be human (huC), as described herein, such as, for example, SEQ ID NO: 28 and SEQ ID NO: 29, respectively. In one embodiment, the TCR alpha constant region and/or the TCR beta chain constant region is as described, for example, in Boulter (2003), Protein Engineering 16, 9: 707-711, particularly in Table I on page 708. The TCR alpha constant region may form one or more cysteine bridges with the TCR beta chain constant region, or vice versa, e.g. contain one or more cysteine residues replacing a serine or threonine residue. obtain. In one embodiment, according to the invention, the TCR alpha chain constant region is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 27. may comprise or consist of an amino acid sequence that is or is the same (preferably is the same). In one embodiment, according to the invention, the TCR beta chain constant region is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 28. may comprise or consist of an amino acid sequence that is or is the same (preferably is the same).

In one embodiment of the invention, the TCR described and provided herein comprising CDR3 according to (A) is
(Aa3) An amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to SEQ ID NO: 20. Contains or consists of
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 47-51 of SEQ ID NO: 20 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 69-75 of SEQ ID NO: 20 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 109-123 of SEQ ID NO: 20 a TCR alpha chain comprising or consisting of the sequence and/or (Ab3) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 22; comprising or consisting of identical (preferably identical) amino acid sequences;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 46-50 of SEQ ID NO: 22 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 68-73 of SEQ ID NO: 22 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 110-122 of SEQ ID NO: 22 TCR beta chain comprising or consisting of a sequence.

In another embodiment of the invention, the TCR described and provided herein comprising CDR3 according to (B) is
(Ba3) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to SEQ ID NO: 48; Contains or consists of
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 45-49 of SEQ ID NO: 48 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 67-73 of SEQ ID NO: 48 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 107-121 of SEQ ID NO: 48 a TCR alpha chain comprising or consisting of the sequence and/or (Bb3) at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to SEQ ID NO: 50; comprising or consisting of identical (preferably identical) amino acid sequences;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 44 to 49 of SEQ ID NO: 50 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 67-71 of SEQ ID NO: 50 contains or consists of an array;
Amino acids that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar or identical (preferably identical) to positions 108 to 122 of SEQ ID NO: 50 TCR beta chain comprising or consisting of a sequence.

In one embodiment of the invention, the TCR described and provided herein is
(A) CDR3 alpha chains, described herein under (Aa), covalently linked to each other to form a TCR heterodimer or multimer, herein described under (Aa1); at least one TCR alpha chain or subregion thereof according to the CDR1/2 alpha chain described under (Aa2), the TCR variable alpha chain described under (Aa3), or the TCR alpha chain described under (Aa3); and the CDR3 beta chain described herein under (Ab), the CDR1/2 beta chain described herein under (Ab1), the TCR variable beta chain described herein under (Ab2). or (B) covalently linked to each other to form a TCR heterodimer or multimer. CDR3 alpha chain as described herein under (Ba), CDR1/2 alpha chain as described herein under (Ba1), TCR variable alpha as described herein under (Ba2) at least one TCR alpha chain or a subregion thereof according to the chain, or TCR alpha chain described under (Ba3), and the CDR3 beta chain, described herein under (Bb), (Bb1 ), a TCR variable beta chain as described herein under (Bb2), or a TCR beta chain as described herein under (Bb3). contains two TCR beta chains or subregions thereof.

According to the present invention, the TCR described and provided herein can be any type of TCR. In one embodiment of the invention, the TCR may be selected from the group consisting of native TCRs, TCR variants, TCR fragments, and TCR constructs. In a preferred embodiment of the invention, the TCR is water soluble.

According to the invention, all TCR variants are preferably functional variants of the TCR of the invention. As used herein, the term "functional variant" has substantial or substantial sequence identity or similarity with the parent TCR, its variable region, or its antigen-binding region and its biological activity. , i.e., the parent TCR of the invention specifically binds to an antigenic target with a similar, the same, or even higher degree of antigenic specificity as the TCRs disclosed herein and evaluated in the accompanying Examples. Refers to a TCR, polypeptide, or protein that shares that ability. TCR sequence variants are also encompassed by the invention.

As used herein, the term "TCR variant" refers to a "sequence variant" of the TCR disclosed herein, provided that the variant preferably retains the antigenic specificity of the "parent" TCR of the present invention. , i.e. substantially comprises the amino acid sequence of the TCR of the invention described above (also referred to as the "parent" TCR), but with at least one amino acid modification (i.e., substitution) compared to the "parent" TCR amino acid sequence. , deletions, or insertions). TCR sequence variants of the invention are typically prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the "parent" TCR or by peptide synthesis. Generally, the aforementioned amino acid modifications may be introduced or present in the variable or constant regions of the TCR, and may affect binding strength and specificity, post-translational processing (e.g., glycosylation), thermodynamic stability, solubility, It may serve to modulate properties such as surface expression or TCR association.

The term "TCR" as used herein further includes TCR constructs. The term "construct" includes at least one antigen-binding domain of a TCR of the invention, but does not necessarily include a variable incorporated into the basic structure of a native TCR (i.e., a TCR alpha chain and a TCR beta chain forming a heterodimer). proteins or polypeptides that do not share a domain). TCR constructs and fragments are typically obtained by routine methods of genetic engineering and are often artificially constructed to contain additional functional protein or polypeptide domains. According to the foregoing, it is envisaged that the TCR constructs and fragments of the invention will include at least one CDR3 alpha and/or at least one CDR3 beta as disclosed elsewhere herein. at least one of CDR1 alpha, CDR2 alpha, CDR1 beta, CDR2 beta, alpha chain variable region, beta chain variable region, alpha chain and/or, optionally in combination with additional protein domains or moieties exemplified herein. or beta chains, or combinations thereof, are further contemplated herein. It is envisioned that the TCR constructs and fragments provided herein are capable of specifically binding the same antigenic targets as the TCRs of the invention described above and evaluated in the accompanying Examples.

The TCRs of the present invention are heterodimers in which at least one TCR alpha chain variable region or TCR alpha chain and at least one TCR beta chain variable region are covalently linked to each other to form a TCR heterodimer or multimer. including bodies and multimers. A "multimer" as used in the present invention describes a molecule of diverse subunits or functional entities, whereas a heterodimer includes only two functional entities. In its simplest form, a multivalent TCR construct according to the invention comprises three or four or It contains a larger number of TCR multimers. In this context, "covalently linked" refers to a chemical bond between two molecules that share a pair of electrons that describes a stable equilibrium between the atomic bonds.

According to the invention, suitable linkers may have spheres, preferably uniform beads, more preferably polystyrene beads, most preferably biocompatible polystyrene beads. Such a TCR construct may also be composed of a TCR of the invention and a bead with a predetermined fluorescent dye incorporated into the bead. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin, and extravidin, each of which has four binding sites for biotin. . Thus, biotinylated TCRs can be formed into multimers with multiple TCR binding sites. The number of TCRs in a multimer will depend on the amount of TCRs in relation to the amount of linker molecule used to create the multimer, and also on the presence or absence of any other biotinylated molecules. Dew. Exemplary multimers are dimers, trimers, tetramers, or pentamers, or higher order multimeric TCR constructs. Multimers of the invention may also contain further functional entities such as labels or drugs or (solid) carriers.

According to the invention, a TCR heterodimer or multimer comprises at least one TCR alpha chain, TCR alpha chain variable region, or CDR3alpha, and/or at least one TCR beta chain, TCR beta chain variable region, or Also concerned are fusion proteins or polypeptides comprising CDR3beta; as well as one or more additional fusion components. It is an antibody or mononucleotide directed against at least one TCR alpha chain as defined herein and/or at least one TCR beta chain as defined herein and/or an antigen or epitope on the surface of a lymphocyte. It may be a chain antibody fragment (scFv), and the TCR alpha chain and TCR beta chain are linked to each other and optionally via a linker, fused to said antibody or scFv. Useful components include Fc receptors; Fc domains (derived from IgA, IgD, IgG, IgE, and IgM); cytokines (such as IL-2 or IL-15); toxins; antibodies or antigen-binding fragments thereof (anti- CD3, anti-CD28, anti-CD5, anti-CD16, or anti-CD56 antibodies, or antigen-binding fragments thereof); CD247 (CD3-zeta), CD28, CD137, CD134 domains; or any combination thereof.

Exemplary antibody fragments that can be used as fusion components according to the invention include (s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "r IgG" ("half-antibody"), etc. , fragments of full-length antibodies; scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's); Modified antibody fragments such as tandem di-scFv, tandem tri-scFv, multibodies such as minibodies, triabodies or tetrabodies; and nanobodies or monobodies containing only one variable domain, which can be VHH, VH, or VL. Includes single domain antibodies, such as single variable domain antibodies.

The TCR constructs of the present invention can be fused to one or more antibodies or antibody fragments, forming monovalent, bivalent, and multivalent/multivalent constructs, thus providing a single antibody that specifically binds only one target antigen. Monospecific constructs, as well as bispecific and multispecific/multispecific, which specifically bind more than one target antigen, e.g., two, three, or more, through separate antigen binding sites. Produces a construct.

Optionally, a linker is provided between one or more of the domains or regions of the TCR constructs of the invention, i.e. TCR alpha chain CDR3, TCR alpha chain variable region, and/or TCR alpha chain, TCR beta chain CDR3. , the TCR beta chain variable region, and/or the TCR beta chain, and/or one or more of the fusion components described herein. Linkers are known in the art and reviewed by, inter alia, Chen et al., Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-1369. Generally, linkers include flexible, cleavable, and rigid linkers and will be selected depending on the type of construct and intended use/application. For example, with respect to therapeutic applications, non-immunogenic, flexible Linkers are often preferred. Such linkers are generally composed of small non-polar (eg, GIy) or polar (eg, Ser or Thr) amino acids, including "GS" linkers consisting of a stretch of GIy and Ser residues.

Particularly useful TCR constructs contemplated according to the invention include at least one antibody or antibody fragment directed to an antigen or epitope on the surface of a lymphocyte, optionally linked to each other and optionally via a linker. at least one TCR alpha chain, TCR alpha chain variable region, or CDR3 alpha, at least one TCR beta chain, as defined herein, fused to a single chain antibody fragment (scFv, etc.) , TCR beta chain variable region, or CDR3beta. Useful antigenic targets recognized by antibodies or antibody fragments (eg, scFv) include CD3, CD28, CD5, CD16, and CD56. The "TCR portion" (i.e., TCR alpha and beta chains, or variable regions or CDR3 thereof) retains its ability to recognize an antigenic target as defined herein, and the "antibody portion" retains the ability to recognize the desired surface antigen or The construct can generally have any structure, so long as it binds to the epitope and thereby directs and targets the respective lymphocyte to the target cell. Such constructs may advantageously serve as "adapters" to join together antigen-presenting cells presenting antigen targets (such as tumor cells) and lymphocytes (such as cytotoxic T cells or NK cells). An example of such a fusion protein is the bispecific T cell engager (BiTE), which consists of two single chain variable fragments (scFv) of various antibodies on a single peptide chain of approximately 55 kilodaltons (kD). This is a construct operated according to the principles of (Registered Trademark)). Accordingly, the TCR constructs of the invention include at least one TCR antigen binding domain as described herein linked to a scFV (or other binding domain) of desired binding specificity, e.g. TCR variable alpha and variable beta chains) fused together. The scFv (or other binding domain) can be directed to T cells, such as through the CD3 receptor, or CD56 for NK cell activation, or to tumors, on the other hand, through antigen targets specifically expressed on tumor cells. Binds to cells. at least one TCR antigen binding domain as described herein, an scFv (or other binding domain), and an additional domain (e.g., an Fc domain) for targeting the construct to a site of action in the body, e.g. Triabodies are also contemplated herein.

TCRs of the invention may be provided in "isolated" or "substantially pure" form. "Isolated" or "substantially pure" as used herein means that the TCR has been identified and isolated and/or recovered from components of its production environment. and thereby an "isolated" TCR is free or substantially free of other contaminating components from its environment of production that could interfere with its therapeutic or diagnostic use. It is. Contaminant components can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Thus, an "isolated" TCR is defined as at least one step that incubates a host cell under conditions that cause expression of said TCR, purifies said TCR, and thus removes or substantially removes these contaminating components. The method for obtaining TCR will be prepared by including a purification step. The foregoing definitions are equally applicable mutatis mutandis to "isolated" polynucleotides/nucleic acids.

TCRs of the invention may be provided in soluble form. Soluble TCRs are useful as diagnostic tools and as carriers or "adapters" to specifically target therapeutic agents or effector cells, eg, to cancer cells expressing the antigenic target recognized by the soluble TCR. Soluble TCRs (sTCRs) are typically stabilized, optionally via disulfide bonds, or covalently linked via suitable linker molecules, e.g. as described above in the context of the TCR constructs of the invention. fragments or constructs comprising the TCR alpha and/or beta chains, or variable regions or CDRs thereof, linked together. They typically will not include transmembrane regions, for example. In some situations, especially when produced in a recombinant host that does not provide the aforementioned attributes, amino acid modifications in the polypeptide sequence may be introduced to improve the solubility of the molecule and/or the correct folding and pairing of the alpha and beta chains (desired case). When using E. coli as the production host cell, for example, folding and pairing of TCR alpha and beta chains is typically accomplished in vitro. Thus, a TCR according to the invention may include, for example, additional cysteine residues, as described elsewhere herein. In a preferred embodiment of the invention, the TCR is water soluble.

Besides additional cysteine bridges, other useful modifications are, for example, Walseng et al., (2015), to increase folding, expression, and/or pairing of TCR alpha and/or beta chains. including the addition of leucine zipper and/or ribosome skipping sequences, such as the 2A sequence from picornaviruses, as described in PLoS ONE 10(4): e0119559.

TCRs of the invention may further include one or more modifications described below. The modifications described below are typically considered covalent modifications and can be accomplished using standard techniques known in the art. In certain situations, amino acid modifications in the TCR may be required to facilitate the introduction of said modifications.

According to the invention, the TCRs described and provided herein include one or more fusion components, e.g., Fc receptors; Fc domains including IgA, IgD, IgG, IgE, and IgM; Cytokines, including IL-2 or IL-15; toxins; antibodies or antigen-binding fragments thereof, including anti-CD3, anti-CD28, anti-CDS, anti-CD16, or anti-CD56 antibodies, or antigen-binding fragments thereof; and CD247 (CD3 - zeta), CD28, CD137, CD134 domains, or combinations thereof; optionally further comprising at least one linker.

In one embodiment of the invention, the TCR described and provided herein is
(A) CDR3 alpha chains as described herein under (Aa), optionally covalently linked to each other to form a TCR heterodimer or multimer; at least one TCR alpha chain or its CDR1/2 alpha chain as described in subregions, and the CDR3 beta chain described herein under (Ab), the CDR1/2 beta chain described herein under (Ab1), and the TCR described herein under (Ab2). at least one TCR beta chain or subregion thereof according to the variable beta chain, or TCR beta chain described under (Ab3), or (B) optionally covalently linked to each other and a TCR heterodimer. CDR3 alpha chain as described herein under (Ba), CDR1/2 alpha chain as described herein under (Ba1), forming a body or a multimer; at least one TCR alpha chain or subregion thereof according to a TCR variable alpha chain as described, or a TCR alpha chain as described under (Ba3), and as described herein under (Bb) CDR3 beta chain, CDR1/2 beta chain described herein under (Bb1), TCR variable beta chain described herein under (Bb2), or TCR beta chain described herein under (Bb3) at least one TCR beta chain or subregion thereof according to
The TCR further comprises an antibody or single chain antibody fragment (scFv) directed against an antigen (e.g., CD3, CD28, CD5, CD16, or CD56) or epitope on the surface of the lymphocyte;
The TCR alpha chain or subregion thereof and the TCR beta chain or subregion thereof are linked to each other and optionally fused to said antibody or scFv via a linker.

The term "epitope" as used herein refers to a site on an antigen that a recognition molecule (eg, a TCR as described and provided herein) binds. Preferably, an epitope is a site on a molecule to which a recognition molecule, preferably a TCR or antibody, will be raised and/or to which a TCR or antibody will bind. For example, an epitope can be recognized by a recognition molecule, particularly preferably by a TCR or an antibody that defines the epitope. A "linear" epitope is an epitope whose primary amino acid sequence includes the recognized epitope. Linear epitopes typically contain at least 3, more usually at least 5, eg, about 8 to about 10 amino acids in unique sequence.

In one embodiment of the invention, the TCRs described and provided herein can further include at least one molecular marker.

TCRs of the invention, particularly (soluble) TCRs, may be labeled with at least one molecular marker. Useful molecular markers are known in the art and can be conjugated to the TCR or TCR variant using routine methods, optionally via linkers of various lengths.

In general, different markers are classified into various classes depending on the assay in which they are detected; Possible isotopic markers (e.g., <3>H, <14>, <15>N, <35>S, <89>Zr, <90>Y, <99>Tc, <111>In, <125> I, <131>I); magnetic markers (e.g., magnetic particles); redox-active moieties; fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and "small molecule" fluorophores. optical dyes such as fluorophores (including, but not limited to, chromophores, phosphors, and fluorophores), which can be either proteinaceous or proteinaceous fluorophores; enzyme groups (e.g., horseradish peroxidase); , β-galactosidase, luciferase, alkaline phosphatase); a biotinylated group; or a predetermined polypeptide recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for a secondary antibody, metal binding domain, epitope tag, etc.); Including, but not limited to, peptide epitopes. If the TCR, TCR variant, or especially soluble TCR construct (such as those containing at least one TCR alpha and/or TCR beta chain as described herein) is intended for diagnostic use, labeling with a molecular marker. It is particularly assumed that

TCRs of the invention, particularly soluble TCRs, e.g. reduce immunogenicity, increase hydrodynamic size (size in solution), solubility, and/or stability (e.g. protect against proteolytic degradation). may be modified by attaching additional functional moieties to increase serum half-life) and/or to extend serum half-life.

Exemplary functional moieties for use in accordance with the invention include peptides or protein domains that bind to other proteins in the human body, such as serum albumin, immunoglobulin Fc regions, or fetal Fc receptors (FcRn). , polypeptide chains of various lengths (e.g. Non-proteinaceous polymers include, but are not limited to, various polyols such as copolymers of carbohydrates such as (eg, HESylation®) or polysialic acid (eg, PolyXen® technology).

Other useful functional moieties include "suicide" or "safety switches" that can be used to shut down effector host cells carrying a TCR of the invention within a patient. An example is the inducible caspase 9 (iCasp9) "safety switch" described by Gargett and Brown Front Pharmacol. 2014; 5: 235. Briefly, effector host cells are modified such that dimerization is dependent on small molecule dimerizing drugs such as AP1903/CIP and express a caspase-9 domain leading to rapid induction of apoptosis in engineered effector cells. is modified by a well-known method. The system is described, for example, in EP-A-2173869. Examples for other "suicide" "safety switches" are known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), expression of CD20 and subsequent depletion using anti-CD20 antibodies, or the myc tag. (Kieback et al., Proc Natl Acad Sci USA. 2008 Jan. 15;105(2):623-8). The TCRs of the invention may also be modified by introducing inducible so-called "on switches" (e.g. as described in WO2019175209), with modified alpha and beta chains of the TCRs of the invention. dimerizes only upon interaction with a small dimerizing drug, resulting in a functional TCR that is subsequently expressed at the cell surface only in the presence of the dimerizing drug.

TCRs with altered glycosylation patterns are also envisioned herein. As is known in the art, glycosylation pattern refers to the amino acid sequence (e.g., the presence or absence of particular glycosylated amino acid residues, discussed below), and/or the host cell or organism in which the protein is produced. may depend on. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Addition of an N-linked glycosylation site to a binding molecule involves selecting an amino acid sequence from asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline. Conveniently this is accomplished by modification to contain one or more tripeptides. O-linked glycosylation sites may be introduced by addition to or replacement of one or more serine or threonine residues to the starting sequence.

Another means of glycosylation of TCRs is through chemical or enzymatic conjugation of glycosides to proteins. Depending on the conjugation mode used, the sugars may include (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) those of serine, threonine, or hydroxyproline. (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.

Similarly, deglycosylation (i.e., the removal of carbohydrate moieties present on the bound molecule) can be performed chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or by employing endo- and exo-glycosidases. can be accomplished enzymatically by

It is also conceivable to add drugs such as small molecule compounds to the TCRs of the invention, particularly soluble TCRs. Linking can be achieved through covalent bonds or non-covalent interactions such as through electrostatic forces. A variety of linkers known in the art may be employed to form drug conjugates.

The TCRs of the present disclosure, particularly soluble TCRs, can be modified to introduce additional domains that aid in the identification, tracking, purification, and/or isolation of the respective molecules (tags). Non-limiting examples of such tags are Myc tag, HAT tag, HA tag, TAP tag, GST tag, chitin binding domain (CBD tag), maltose binding protein (MBP tag), Flag tag, Strep tag and its peptide motifs known as variants (eg, Strep II tag), His tag, CD20, Her2/neu tag, myc tag, FLAG tag, T7 tag, HA (hemagglutinin) tag, or GFP tag.

Epitope tags are useful examples of tags that can be incorporated into the TCRs of this disclosure. Epitope tags allow binding of specific antibodies and therefore identification and tracking of binding and translocation of soluble TCR or host cells within the patient or in cultured (host) cells. It is a short stretch of amino acids. Detection of epitope tags, and thus tagged TCRs, can be accomplished using several different techniques. Examples of such techniques include immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("Western"), and affinity chromatography. The epitope tag may have a length of, for example, 6 to 15 amino acids, especially 9 to 11 amino acids. It is also possible to include more than one epitope tag in a TCR of the invention.

Tags can be further employed in the stimulation and expansion of host cells bearing TCRs of the invention by culturing the cells in the presence of binding molecules (antibodies) specific for said tags.

The invention further relates to nucleic acids encoding the TCRs described and provided herein. In a specific embodiment of the invention, such a nucleic acid molecule is at least 80% covalent with a nucleic acid sequence of any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21. , 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical; or of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49. Can include nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one nucleic acid sequence.

As used herein, unless specifically defined otherwise, the terms "nucleic acid" or "nucleic acid molecule" are used synonymously with "oligonucleotide," "nucleic acid strand," etc., and include one, It refers to a polymer containing two or more nucleotides, eg single-stranded or double-stranded.

Generally, as used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" should be interpreted interchangeably. Generally, nucleic acid molecules include DNA molecules (dsDNA, ssDNA, cDNA, etc.), RNA molecules (dsRNA, ssRNA, mRNA, ivtRNA, etc.), oligonucleotide thiophosphates, substituted ribo-oligonucleotides, or PNA molecules, among others. may be included. Additionally, the term "nucleic acid molecule" can refer to DNA or RNA or a hybrid thereof, or any modification thereof known in the art (for examples of modifications, see, e.g., U.S. Pat. No. 5,525,711; See patent no. 4,711,955, US patent no. 5,792,608, or European patent application no. 302,175). Polynucleotide sequences can be single-stranded or double-stranded, linear or circular, natural or synthetic, and without any size limitations. For example, the polynucleotide sequence can be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozyme RNA, or DNA encoding such RNA, or a chimeroplast (Gamper, Nucleic Acids Research, 2000 , 28, 4332 - 4339). Said polynucleotide sequence may be in the form of a vector, a plasmid, or in the form of viral DNA or RNA. Also described herein are nucleic acid molecules that are complementary to the nucleic acid molecules described above, and that can hybridize to the nucleic acid molecules described herein. The nucleic acid molecules described herein may also be fragments of nucleic acid molecules in the context of the present invention. In particular, such fragments are functional fragments. An example for such a functional fragment is a nucleic acid molecule that can serve as a primer.

The invention further relates to vectors containing the nucleic acid molecules described and provided herein.

The term "vector" as used herein refers to, among others, plasmids, cosmids, viruses, bacteriophages, and other vectors commonly used in genetic engineering. In one embodiment of the invention, the vector is a host cell described herein, e.g., prokaryotic cells (e.g., (e)bacteria, archaea), eukaryotic cells (e.g., mammalian cells, insect cells), Suitable for transformation, transduction and/or transfection of fungal cells, yeast etc. Examples of bacterial host cells in the context of the present invention include Gram-negative and Gram-positive cells. Preferably, the host cell is a eukaryotic cell, such as a human cell. Specific examples for suitable host cells include lymphoblastoid cell lines, cytotoxic T lymphocytes (CTLs), CD8+ T cells (preferably autologous CD8+ cells), CD4+ T cells (preferably autologous CD4+ cells), among others. cells), T memory stem cells ( _TSCM ), natural killer (NK) cells (e.g. CD3 (CD3 gamma, CD3 delta, CD3 epsilon) as also described and provided in WO 2016/116601). recombinantly modified to express (including)), natural killer T (NKT) cells, and gamma/delta-T cells. In one embodiment of the invention, said vector is suitable for stable transformation of host cells.

Accordingly, in one embodiment of the invention, the vectors provided are expression vectors. Generally, expression vectors are widely described in the literature. Typically, they may contain not only a selectable marker gene and an origin of replication ensuring replication in the selected host, but also a promoter and, in most cases, termination signals for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or polylinker that allows insertion of the nucleic acid sequence/molecule desired to be expressed. It is understood that if the vector provided herein is created by taking advantage of expression vectors known in the prior art, it already contains a suitable promoter to be employed in the context of the present invention. It should be. The nucleic acid construct is preferably inserted into the vector in such a way that the resulting vector contains only one promoter suitable to be employed in the context of the present invention. A person skilled in the art knows how such an insertion can be practiced. For example, the promoter can be excised from the nucleic acid construct or from the expression vector prior to ligation. In one embodiment of the invention, the vector is capable of integrating into the host cell genome. The vector may be any vector suitable for the respective host cell, preferably an expression vector. In the context of the present invention, preferred vectors include lentiviral and retroviral vectors known in the art.

In addition to origins of replication, selectable markers, and restriction enzyme cleavage sites, expression vectors typically include one or more regulatory sequences operably linked to the heterologous polynucleotide to be expressed.

The term "regulatory sequences" refers to nucleic acid sequences necessary for the expression of an operably linked coding sequence of a (heterologous) polynucleotide in a particular host organism or host cell, and thus includes transcriptional and translational control sequences. Typically, the regulatory sequences required for expression of a heterologous polynucleotide sequence in prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. In eukaryotes, a promoter, polyadenylation signal, enhancer, and optionally a splice signal are typically required. Additionally, specific initiation and secretion signals may also be introduced into the vector to enable secretion of the polypeptide of interest into the culture medium.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, particularly on the same polynucleotide molecule. For example, a promoter is operably linked to a coding sequence of a heterologous gene if it is capable of effecting expression of that coding sequence. A promoter is typically placed upstream of a gene encoding a polypeptide of interest and regulates the expression of said gene.

Exemplary regulatory sequences for mammalian host cell expression include cytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus 40 (SV40) (such as the SV40 promoter/enhancer), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and viral elements that direct high-level protein expression in mammalian cells, such as polyoma-derived promoters and/or enhancers. As specified above, the expression vector may also include an origin of replication and a selectable marker.

Particularly useful according to the invention are retroviral and lentiviral vectors. Examples for suitable expression vectors include viral vectors, such as lentiviral or retroviral vectors, such as the MP71 vector or retroviral SIN vector; and lentiviral vectors or lentiviral SIN vectors. It is envisaged that a viral vector containing a polynucleotide encoding a TCR of the invention can, for example, infect lymphocytes, which subsequently express the heterologous TCR. Another example for a suitable expression vector is the SB DNA plasmid of the Sleeping Beauty (SB) transposon transposase DNA plasmid system. Nucleic adds and/or in particular expression constructs of the invention may also be introduced into cells by transient RNA transfection.

Viral vectors currently used for native TCR expression typically contain the TCR-alpha and TCR-beta chain genes in one vector and the internal ribosome entry site (IRES) sequence or 2A derived from porcine tesiovirus. peptide sequences, resulting in the expression of a single messenger RNA (mRNA) molecule under the control of a viral promoter in the transduced cell.

The invention further relates to a host cell comprising a TCR as described and provided herein, a nucleic acid molecule as described and provided herein, or a vector as described and provided herein. A variety of host cells can be used in accordance with the present invention. As used herein, the term "host cell" can be or is a recipient of a polynucleotide or vector described herein and/or expresses a TCR of the invention. (and optionally secreting) cells. The terms "cell" and "cell culture" are used interchangeably to refer to a source of TCR, unless it is explicitly specified otherwise. The term "host cell" also includes host cell lines. In general, the term "host cell" includes prokaryotic or eukaryotic cells, including, without limitation, bacteria, yeast cells, fungal cells, plant cells, and insect and mammalian cells such as mouse, rat, macaque, etc. , or animal cells such as human cells. Thus, the present invention provides, among other things, a host cell comprising a polynucleotide or vector, eg, an expression vector comprising a nucleotide sequence encoding a TCR or TCR construct as described herein. Polynucleotides and/or vectors of the invention can be introduced into host cells using routine methods known in the art, eg, by transfection, transformation, etc.

"Transfection" is the process of intentionally introducing a nucleic acid molecule or polynucleotide (including a vector) into a target cell. An example is RNA transfection, ie the process of introducing RNA (in vitro transcribed RNA, ivtRNA, etc.) into a host cell. The term is mostly used for non-viral methods in eukaryotic cells. The term "transduction" is often used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves creating a transient pore or "hole" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell compaction, or by mixing the material with cationic lipids to produce liposomes that fuse with the cell membrane and deposit their cargo inside. can be executed. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA coprecipitation), microinjection, and electroporation. include.

"Transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and also into non-animal eukaryotic cells, including plant cells. Transformation is therefore a genetic modification of a bacterial or non-animal eukaryotic cell resulting from direct uptake through the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be brought about by artificial means. For transformation to occur, the cells or bacteria must be in a state of competence, which can occur as a time-limited response to environmental conditions such as starvation and cell density. For prokaryotic transformation, techniques can include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation. Techniques for plant transformation are described by A. Agrobacterium-mediated transfer, rapidly propelled tungsten or gold microprojectile, electroporation, microinjection, and polyethylene glycol-mediated uptake, such as by A. tumefaciens.

According to the invention, with respect to the expression of the TCR of the invention, the expression of the inserted polynucleotide sequence is modulated and/or the gene product (i.e. RNA and/or protein) is modified and processed as desired. A host cell can be selected that will Such modifications (eg, glycosylation) and processing (eg, cleavage) of gene products may be important for TCR function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the product. For this purpose, eukaryotic host cells that possess cellular machinery for proper processing of primary transcripts, glycosylation and phosphorylation of gene products may be used.

According to the invention, a host cell comprising a TCR as described and provided herein, a nucleic acid molecule as described and provided herein, or a vector as described and provided herein is It can be any cell suitable for stably expressing the TCRs described and provided herein. Preferably, such a host cell is capable of displaying such a TCR on its surface and contains a PRAME _LL -peptide (or a portion thereof, or a portion thereof, as described herein and specified). and in its specified HLA-A bound form).

A host cell according to the invention may be a "production host cell" used for the expression of a soluble TCR of the invention, and is preferably capable of expressing high amounts of recombinant protein. According to the foregoing, possible expression systems (i.e., host cells containing the expression vectors described above) include bacteria (e.g., E. coli) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors. E. coli, B. subtilis); yeast transformed with recombinant yeast expression vectors (e.g., Saccharomyces, Pichia); recombinant viral expression vectors (e.g., baculovirus) insect cell systems infected with; plant cell systems infected with recombinant viral expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmids), etc. Contains microorganisms. Promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or promoters derived from mammalian viruses (e.g. adenovirus late promoter; vaccinia virus 7.5K promoter, cytomegalovirus (CMV) major immediate early promoter (MIEP) promoter) ) is often preferred. Suitable mammalian host cells may be selected from known cell lines (e.g. COS, CHO, BLK, 293, 3T3 cells), but include cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells. It is also conceivable to use lymphocytes such as , natural killer (NK) cells, natural killer T (NKT) cells, gamma/delta-T cells, etc.

Exemplary mammalian host cells that may be used as "production host cells" include CHO (CHO cells), NSO, COS (SV40 T antigen-producing cells), including DHFR-negative Chinese hamster ovary (CHO) cells such as DG44 and DUXBI 1. (derived from CVI), HEK293 (human kidney), and SP2 (mouse myeloma) cells. Other exemplary host cell lines are HELA (human cervical carcinoma), CVI (monkey kidney line), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast), BALBC. /3T3 (mouse fibroblasts), HAK (hamster kidney line), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells), and RAJI (human lymphocytes); It is not limited. Host cell lines are typically available from commercial services, the American Type Culture Collection (ATCC), or from published literature. Non-mammalian cells such as bacterial, yeast, insect, or plant cells are also readily available and may also be used as "production host cells" as described above. Exemplary bacterial host cells include Enterobacteriaceae, such as Escherichia coli, Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus. (Streptococcus); and Haemophilus influenza. Other host cells include yeast cells such as Saccharomyces cerevisiae and Pichia pastoris. Insect cells include, without limitation, Spodoptera frugiperda cells.

According to the foregoing, the present invention provides methods herein comprising: (a) incubating a host cell (i.e., a production host cell) under conditions that cause expression of a TCR; and (b) purifying said TCR. Also provided are methods for producing and obtaining the TCRs described in the book.

Host cells carrying the expression vectors are grown under conditions suitable for the production of the TCRs provided herein, particularly the alpha and/or beta chains described elsewhere herein, and are grown to produce alpha and/or beta chains as described elsewhere herein. /or assayed for beta chain protein synthesis. For expression of double-stranded TCRs, vectors encoding both alpha and beta chains can be coexpressed in a host cell for expression of the entire molecule. Once the TCR of the invention is expressed, it can be purified by any purification method known in the art, such as by chromatography (e.g., ion exchange chromatography (e.g., hydroxylapatite chromatography), affinity chromatography, especially protein A , Protein G, or lectin affinity chromatography, sizing column chromatography), centrifugation, differential solubility, hydrophobic interaction chromatography, or by any other standard technique for the purification of proteins. One skilled in the art will readily be able to select an appropriate purification method based on the particular characteristics of the TCR to be recovered.

The host cells described and provided in the context of the present invention may also be "effector host cells" containing the nucleotide sequences, vectors, or TCRs of the present invention. It is envisaged that said effector host cells are engineered using routine methods to contain a nucleic acid sequence encoding a TCR of the invention and express the TCR described herein, particularly on the cell surface. For the purposes of the present invention, a "modified host cell expressing a TCR of the invention" generally refers to a cell that has been treated to express a TCR according to the invention, for example by RNA transfection as described in the accompanying Examples. (effector or producer) host cell. Other methods of modification or transfection or transduction are also envisioned, such as those described elsewhere herein. Thus, the term "modified host cell" includes "transfected," "transduced," and "genetically engineered" host cells that preferably express a TCR of the invention. Preferably, such "(modified) effector host cells" (particularly "(modified) effector lymphocytes") are capable of mediating effector functions through intracellular signal transduction upon binding of the TCR to its specific antigen target. It is possible. Such effector functions include, for example, the release of perforin (which creates holes in target cell membranes), granzymes (which are proteases that act within cells to induce apoptosis), and Fas ligand (which induces apoptosis in target cells with FAS). activating) and release of cytokines, preferably Th1/Tc1 cytokines such as IFN-gamma, IL-2, and TNF-α. It is therefore envisaged that an effector host cell engineered to express a TCR of the invention capable of recognizing and binding its antigenic target in the subject to be treated will perform the effector functions described above, thereby Kill target (eg, cancer) cells. Cytolysis of target cells is assessed using the CTL fluorescence killing assay (CTL, USA), which detects the disappearance of fluorescently labeled target cells during co-culture with e.g. TCR-transfected recipient T cells. obtain.

In view of the above, the effector host cell includes a functional TCR, i.e., a functional TCR that typically includes the TCR alpha and beta chains described herein; and also the signal transducing subunits CD3 gamma, delta, epsilon, and zeta (CD3 complex). Additionally, expression of the co-receptors CD4 or CD8 may also be desired. Generally, lymphocytes, T cells, carrying the required genes involved in antigen binding, receptor activation, and downstream signaling (e.g., Lck, FYN, CD45, and/or Zap70) are used as effector host cells. Particularly appropriate. However, effector host cells expressing the TCR of the invention as a "binding domain" without the CD3 signal transducing subunit and/or the aforementioned downstream signaling molecules (i.e., not capable of recognizing the antigenic targets described herein) but do not cause the functions mediated by CD3 and/or the aforementioned downstream signaling molecules) are also contemplated herein. Such effector cells may recognize the antigenic targets described herein and may optionally produce other functions unrelated to CD3 signaling and/or signaling of the aforementioned downstream signaling molecules. It is assumed that Examples include NK or NKT cells expressing a TCR of the invention, which can, for example, release cytotoxic granules upon recognition of their antigenic target.

Therefore, cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, and gamma/delta-T cells are useful lymphocyte effector host cells. considered to be. Such lymphocytes expressing recombinant TCRs of the invention are also referred to herein as "modified effector lymphocytes." However, one skilled in the art will generally appreciate that any component of the TCR signaling pathway that leads to the desired effector function can be introduced into suitable host cells by recombinant genetic engineering methods known in the art. would easily approve. Effector host cells, particularly lymphocytes such as T cells, can be autologous host cells obtained from the subject to be treated and transformed or transduced to express a TCR of the invention. Typically, recombinant expression of TCR will be accomplished by using the viral vectors described in the accompanying Examples. Techniques for obtaining and isolating cells from patients are known in the art.

As previously mentioned, the effector host cells provided herein are specifically contemplated for therapeutic applications. Further genetic modification of host cells may be desirable to increase therapeutic efficacy. For example, when using autologous CD8+ T cells as "effector host cells", suitable additional modifications include down-regulation of endogenous TCR, CTLA-4, and/or PD-1 expression; and/or CD28, CD134 , including the amplification of costimulatory molecules such as CD137. Means and methods for achieving the aforementioned genetic modifications have been described in the art.

Methods for targeted genome manipulation of host cells are known in the art and, in addition to gene knockdown using siRNA, are reviewed in Kim & Kim Nature Reviews Genetics 15, 321-334 (2014), among others. RNA guides derived from zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the bacterial clustered regularly spaced short palindromic repeat (CRISPR)-Cas (CRISPR-related) system. This includes the use of so-called "programmable nucleases" such as engineered nucleases (RGEN). For example, programmable nucleases such as TALENs can be employed to excise DNA regions encoding "unwanted" proteins such as PD-1, CTLA-4, or endogenous TCRs, thereby reducing their expression. . When T cells are used as (effector) host cells, downregulation of endogenous TCRs has the benefit of reducing unnecessary "mispairing" of endogenous and exogenous TCR alpha/beta chains.

In certain embodiments of the invention, such host cells include, for example, lymphoblastoid cell lines, cytotoxic T lymphocytes (CTLs), CD8+ T cells (preferably autologous CD8+ cells), CD4+ T cells (preferably autologous CD4+ cells), T memory stem cells ( _TSCM ), natural killer (NK) cells (e.g. CD3 (CD3 gamma, CD3 delta, CD3 epsilon (including CD3 delta, CD3 epsilon)), natural killer T (NKT) cells, and gamma/delta-T cells.

The invention includes the steps of incubating the host cells described and provided herein under conditions that cause expression of the TCR, and purifying the TCR. The method further relates to a method for obtaining said TCR.

The present invention
(i) TCRs described and provided herein;
(ii) the nucleic acid molecules described and provided herein;
(iii) a vector as described and provided herein; and/or (iv) one or more of the host cells described and provided herein, and optionally a pharmaceutical excipient. It further relates to pharmaceutical or diagnostic compositions comprising:

The term "pharmaceutical composition" specifically refers to a composition suitable for administration to humans. However, compositions suitable for administration to non-human animals are also generally encompassed by the term.

Pharmaceutical compositions envisioned by the present invention further comprise one or more checkpoint inhibitors, preferably selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. obtain. All of the aforementioned inhibitors are immune checkpoint inhibitors capable of down-regulating immune responses. Cytotoxic lymphocyte-associated protein 4 (CTLA-4) inhibitor is a constitutively expressed protein receptor on regulatory T cells, but is upregulated on conventional T cells only after activation. PD-1 and PD-L1 inhibitors act to inhibit the association of programmed death ligand 1 (PD-L1) with its receptor programmed cell death protein 1 (PD-1). Interactions of these cell surface proteins are involved in the suppression of the immune system and occur after infection to limit killing of bystander host cells and prevent autoimmune diseases. Therefore, it is preferred that said checkpoint inhibitor is combined in a pharmaceutical composition according to the invention.

According to the invention, the pharmaceutical compositions described and provided herein can further include a checkpoint inhibitor. In one embodiment of the invention, said checkpoint inhibitor may be selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.

Additional checkpoint inhibitors encompassed by the invention are LAG3, ICOS, TIM3, VISTA, and CEACAM1. LAG3 is an inhibitory receptor on antigen-activated T cells. ICOS proteins belong to the CD28 and CTLA-4 cell surface receptor families. It forms homodimers and plays important roles in the regulation of cell-cell signaling, immune responses, and cell proliferation. TIM3 or Hepatitis A Virus Cellular Receptor encodes a protein that belongs to the immunoglobulin superfamily and the TIM family of proteins. CD4-positive T helper lymphocytes can be divided into types 1 (Th1) and 2 (Th2) based on their cytokine secretion patterns. VISTA or V-Set Immunoregulatory Receptor encodes an immunoregulatory receptor that inhibits T cell responses. The CEACAM1 gene encodes a member of the carcinoembryonic antigen (CEA) gene family, which belongs to the immunoglobulin superfamily. These checkpoint inhibitors may also be combined in pharmaceutical compositions.

The pharmaceutical composition and its components (i.e., the active agent and optionally the excipient) are preferably pharmaceutically acceptable, i.e., capable of producing the desired amount without causing any undesirable local or systemic effects in the recipient. can bring out the therapeutic effects of Pharmaceutically acceptable compositions of the invention may be sterile, for example. Specifically, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally accepted pharmacopoeia for use in animals, and more particularly in humans. obtain.

The active agents described above (eg, host cells or TCR) are preferably present in the pharmaceutical composition in a therapeutically effective amount. By "therapeutically effective amount" is meant an amount of active agent that elicits the desired therapeutic effect. Therapeutic efficacy and toxicity are determined by standard procedures, e.g. ED ₅₀ (dose therapeutically effective in 50% of the population) and LD ₅₀ (dose lethal to 50% of the population), e.g. in cell culture or in test animals. can be done. The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio ED ₅₀ /LD ₅₀ . Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

Precise dosages of TCR polynucleotides, vectors, or host cells will be ascertainable by those skilled in the art using known techniques. A suitable dosage will provide a sufficient amount of the active agent of the invention to be preferably therapeutically effective, ie, to elicit the desired therapeutic effect.

As is known in the art, the purpose of treatment (e.g., maintenance of disease remission versus acute flare-up), route, time and frequency of administration, time and frequency of administration formulation, age, weight, general health, gender. Adjustments to dietary habits, severity of disease state, drug combinations, response sensitivity, and tolerability/response to therapy may be necessary. Suitable dosage ranges for, for example, soluble TCRs described herein can be determined using data obtained from cell culture assays and animal studies, and can include the _ED50 . Typically, dosages may vary from 0.1 to 100,000 micrograms, up to about 2 g total dose, depending on the route of administration. Exemplary dosages of active agents of the invention are about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg kg to about 5 mg/kg, about 0.01 mg/kg to about 1 mg/kg, or about 0.1 mg/kg to about 1 mg/kg. Guidance regarding specific dosages and methods of delivery is provided in the literature. It is recognized that treatment may require the administration of a single therapeutically effective dose, or multiple administrations of therapeutically effective doses of an active agent of the invention. For example, some pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks, or within a month, depending on the formulation, half-life, and clearance rate of the particular composition. Can be administered once. As specified above, the pharmaceutical composition may optionally contain one or more excipients and/or additional active agents.

The term "excipient" includes fillers, binders, disintegrants, coatings, adsorbents, anti-adhesives, lubricants, preservatives, antioxidants, flavors, colors, sweeteners, solvents, co-solvents, Includes buffering agents, chelating agents, viscosity-imparting agents, surfactants, diluents, humectants, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity adjusters. The selection of appropriate excipients for preparing the desired pharmaceutical compositions of the invention is within the knowledge of those skilled in the art. Exemplary carriers for use in the pharmaceutical compositions of the invention include saline, buffered saline, dextrose, and water. Typically, the selection of a suitable excipient will depend, among other things, on the active agent used, the disease to be treated, and the desired formulation of the pharmaceutical composition.

The present invention provides one or more of the active agents of the invention (e.g. host cells or TCR constructs) specified above, as well as one or more active agents suitable for the treatment and/or prevention of the disease to be treated. Further provided are pharmaceutical compositions containing additional active agents. Preferred examples of active ingredients suitable for combination are cis-platin, maytansine derivatives, raschelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II. II), known anticancer drugs such as temozolmide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin; and ricin, diphtheria toxin, pseudomonas bacterial exotoxin Peptide cytotoxins such as A, DNAase, and RNAase; radionuclides such as iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213; prodrugs such as antibody-directed enzyme prodrugs. ; immunostimulants such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulating protein, antibodies or fragments thereof such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains; , homologous protein domains, viral/bacterial protein domains, and viral/bacterial peptides.

A variety of routes are applicable for administering the pharmaceutical composition according to the invention. Typically, administration will be accomplished parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

The pharmaceutical compositions of the invention may be formulated in a variety of forms, depending inter alia on the active agent used (e.g. soluble TCR), e.g. in solid, liquid, gaseous or lyophilized form, inter alia in ointments, creams, In the form of transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or as desired for administration. may be in a form that is particularly suitable for the method of Processes known per se for producing medicaments are presented in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), such as conventional mixing, dissolving, granulating, dragee-coating. It may include production, levigating, emulsification, encapsulation, encapsulation, or lyophilization steps. Pharmaceutical compositions described herein, including, for example, host cells or soluble TCRs, will typically be provided in liquid form and will preferably include a pharmaceutically acceptable buffer.

After the pharmaceutical compositions of the invention have been prepared, they can be placed in a suitable container and labeled for the treatment of the indicated condition. Such labeling would include, for example, amount, frequency, and method of administration. In view of the foregoing, the present invention therefore provides the TCRs, nucleic acids, vectors, and/or as described herein for use as medicaments in the detection, diagnosis, prognosis, prevention, and/or treatment of cancer. or provide host cells.

TCRs, nucleic acids, vectors, and/or host cells generally can be employed for therapeutic detection, diagnosis, prognosis, prevention, and/or treatment of diseases or disorders. The term "treatment" in all its grammatical forms includes therapeutic or prophylactic treatment of a subject in need thereof. "Therapeutic or prophylactic treatment" means preventive treatment aimed at complete prevention of clinical and/or pathological signs, or therapeutic treatment aimed at amelioration or remission of clinical and/or pathological signs; including. Thus, the term "treatment" also includes amelioration or prevention of disease.

Such diseases envisaged to be treated when using the pharmaceutical composition of the present invention are preferably melanoma, bladder carcinoma, colon carcinoma, and breast cancer, sarcoma, prostate cancer, uterine cancer, Membranous cancer, uveal melanoma, squamous cell head and neck cancer, synovial carcinoma, Ewing's sarcoma, triple negative breast cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin cancer Consists of lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct carcinoma, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia. Preferably the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, or colorectal cancer, sarcoma or osteosarcoma.

The terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein to refer to any subject, particularly a mammalian subject, for whom therapy is desired. Mammalian subjects generally include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, and the like. However, it will be readily appreciated that the TCRs, nucleic acids, vectors, host cells, and pharmaceutical compositions provided herein are particularly contemplated for the treatment of human subjects, particularly those who are HLA-A2 positive. Will.

For therapy, a TCR of the invention, particularly a soluble TCR of the invention, a nucleic acid, a vector (such as a viral vector), or a host cell, can be administered directly to a subject in need thereof. Thus, the present invention provides TCRs, nucleic acids, vectors, or host cells for use in methods of cancer detection, diagnosis, prognosis, prevention, and/or treatment. The method provides one or more of (a) (i) a TCR, (ii) a nucleic acid, (iii) a vector, (iv) a host cell, and/or (v) a pharmaceutical composition of the invention. and (b) administering one or more of (i) to (v) to a subject in need thereof. Optionally, the method may include further steps of cancer therapy, such as radiation, or administration of one or more anti-cancer agents.

Treatment according to the invention includes the steps of: (a) providing a sample of a subject, said sample comprising lymphocytes; (b) (i) a TCR of the invention; (ii) a nucleic acid; (ii) ) providing one or more of a vector, (iv) a host cell, and/or (v) a pharmaceutical composition; (c) providing the lymphocytes of step (a) with (i) of step (b); (d) administering the modified lymphocytes of step (c) to a subject or patient in need thereof; may be included. It is particularly envisaged that the lymphocytes provided in step (a) are "effector host cells" as described above, advantageously T cells, NK cells and/or NKT cells, especially CD8 ⁺ T cells. may be obtained in the previous step from a sample of the subject, particularly a blood sample, by routine methods known in the art. However, it is also conceivable to use other lymphocytes, preferably capable of expressing the TCR of the invention and exerting the desired biological effector functions described herein. Furthermore, the lymphocytes will typically be selected for compatibility with the subject's immune system, ie they will preferably not elicit an immunogenic response. For example, it is conceivable to use "universal recipient cells", ie, universally compatible lymphocytes that can be grown and expanded in vitro and that exert the desired biological effector functions. The use of such cells would therefore obviate the need to obtain and provide the subject's own lymphocytes in step (a). The ex vivo introduction of step (c) is carried out by introducing the nucleic acids or vectors described herein by electroporation into lymphocytes, or by introducing lentiviral or retroviral vectors as previously described in the context of effector host cells. This can be carried out by infecting lymphocytes with a viral vector such as . Other possible methods include the use of transfection reagents such as liposomes, or transient RNA transfection. Transfer of antigen-specific TCR genes into (primary) T cells, for example by (retro)viral vectors or transient RNA transfection, to generate tumor-associated antigen-specific T cells that can later be reintroduced into the donor. are promising tools in which they specifically target and destroy tumor cells expressing said antigens. In the present invention, said tumor-associated antigen is PRAME as defined herein, especially in its HLA-A ^* 24 or HLA-A ^* 02:17 binding form.

Treatment according to the invention may also include the step of: (a) providing a sample of a subject, said sample comprising lymphocytes; while treatment comprises (b) (i) TCR; (ii) (iii) a vector; (iv) a host cell; and (v) a pharmaceutical composition; (c) providing the lymphocytes of step (i) of step (b); (d) administering the modified lymphocytes of step (c) to a subject or patient in need thereof; Become.

In view of the above, a further aspect of the invention is therefore the use of the TCRs, nucleic acid sequences, vectors, and/or host cells described elsewhere herein to generate modified lymphocytes. be. Means and methods for introducing eg nucleic acids and vectors into lymphocytes are described elsewhere herein.

The invention also provides diagnostic compositions comprising the TCRs, nucleic acids, vectors, and/or host cells described herein as one or more diagnostic agents. Typically, said diagnostic agent will include a means for detecting its binding to its antigenic target, such as a label as described in the context of the TCR constructs of the invention. Regarding host cells, it is conceivable to use modified host cells which contain, for example, dyes or contrast agents that are released upon antigen recognition (instead of cytotoxic granules).

The present invention relates to the TCRs described and provided herein, the nucleic acid molecules described and provided herein, the vectors described and provided herein, and and/or further to the host cells described and provided herein.

The present invention relates to the TCRs described and provided herein, the nucleic acid molecules described and provided herein, the nucleic acid molecules described and provided herein, for use in the detection, diagnosis, prognosis, prevention, and/or treatment of cancer. Further directed to the vectors described and provided herein and/or the host cells described and provided herein. In the context of the present invention, in specific embodiments, cancer is melanoma, bladder carcinoma, colon carcinoma, breast carcinoma, sarcoma, prostate cancer, uterine cancer, uveal cancer, uveal melanoma, squamous head and neck carcinoma. , synovial carcinoma, Ewing's sarcoma, triple negative breast cancer, thyroid cancer, testicular cancer, renal cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), non-Hodgkin's lymphoma, multiple Sexual myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct carcinoma, breast cancer, bladder cancer, myeloid leukemia, acute lymphoblastic leukemia, acute lymphocytic cancer , acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain tumor, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile ducts, cancer of the joints, neck, gallbladder or cancer of the pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, Gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharynx cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, gastrointestinal cancer, testicular cancer , thyroid cancer, uterine cancer, ureteral cancer, and urinary bladder cancer.

According to the invention, in one embodiment, the prevention and/or treatment of cancer comprises:
(i) a TCR as described and provided herein;
(ii) a nucleic acid molecule as described and provided herein;
(iii) the vectors described and provided herein;
(iv) providing one or more of the host cells described and provided herein; and (v) the pharmaceutical compositions described and provided herein; The method may include administering at least one of (i) to (v) to a subject.

According to the invention, in another embodiment, the prevention and/or treatment of cancer comprises:
(1) providing a sample of a subject, the sample comprising lymphocytes;
(2)
(i) a TCR as described and provided herein;
(ii) a nucleic acid molecule as described and provided herein;
(iii) the vectors described and provided herein;
(iv) providing one or more of the host cells described and provided herein; and (v) the pharmaceutical compositions described and provided herein;
(3) introducing one or more of (i) to (v) of step (2) into the lymphocytes of step (1), thereby obtaining modified lymphocytes; and (4) introducing the modified lymphocytes. The method may include administering the modified lymphocytes of step (3) to a subject or patient in need thereof.

The present invention
providing a sample of interest, the sample comprising one or more cells;
The sample;
(i) a TCR as described and provided herein;
(ii) contacting a host cell as described and provided herein, and/or (iii) a pharmaceutical composition as described and provided herein, thereby forming a complex; and The method further relates to a method of detecting the presence of cancer in a subject in vitro comprising the step of detecting a complex, wherein the detection of the complex is indicative of the presence of cancer in the subject.

The present invention relates to the TCRs described and provided herein, the nucleic acid molecules described and provided herein, and/or the nucleic acid molecules described and provided herein for producing modified lymphocytes. further relates to the use of vectors.

The present invention can also be characterized by the following items.
1. A polypeptide comprising an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), or a part of said polypeptide, or each of said polypeptide or a part thereof, in which not more than 1.4 amino acids have been substituted. A T cell receptor (TCR) capable of binding to HLA-A bound forms,
(A)
(Aa) CDR3 of the TCR alpha chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 12, and/or (Ab) CDR3 of the TCR beta chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 14; or (B)
(Ba) CDR3 of the TCR alpha chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 40, and/or (Bb) CDR3 of the TCR beta chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 42.
Including TCR.
2. The TCR comprising CDR3 according to (A) is
(Aa1) CDR1 of a TCR alpha chain comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 4, and/or a TCR alpha comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 8; CDR2 of the TCR beta chain, and/or (Ab1) at least 80% similar to the amino acid sequence of SEQ ID NO: 10; CDR2 of the TCR beta chain containing the amino acid sequence
or comprising a CDR3 according to (B),
(Ba1) CDR1 of a TCR alpha chain comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 32, and/or a TCR alpha comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 36; CDR2 of the TCR beta chain, and/or (Bb1) at least 80% similar to the amino acid sequence of SEQ ID NO: 38; CDR2 of the TCR beta chain containing the amino acid sequence
The TCR according to item 1, further comprising:
3. The TCR according to item 1 or 2, wherein HLA-A is an HLA-A ^* 24 or HLA-A ^* 02 encoding molecule.
4. A TCR according to any one of items 1 to 3, wherein binding of the TCR to the polypeptide or a portion thereof, or an HLA-A bound form thereof, induces IFN-gamma secretion by a cell containing the TCR. .
5. Said induction of IFN-gamma secretion of a cell comprising said TCR to or to a polypeptide comprising an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), in which not more than 4 amino acids have been substituted. or to the respective HLA-A bound form of said polypeptide or a portion thereof is at least 5-fold higher compared to control cells without said TCR.
6. The TCR comprising CDR3 according to (A) is
(Aa2) comprises an amino acid sequence that is at least 80% similar to SEQ ID NO: 16;
comprising an amino acid sequence that is at least 80% similar to positions 47-51 of SEQ ID NO: 16;
comprising an amino acid sequence that is at least 80% similar to positions 69-75 of SEQ ID NO: 16;
a TCR alpha chain variable region comprising an amino acid sequence at least 80% similar to positions 109-123 of SEQ ID NO: 16, and/or (Ab2) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 18;
comprising an amino acid sequence that is at least 80% similar to positions 46 to 50 of SEQ ID NO: 18;
comprising an amino acid sequence that is at least 80% similar to positions 68-73 of SEQ ID NO: 18;
said TCR comprising a TCR beta chain variable region comprising an amino acid sequence at least 80% similar to positions 110-122 of SEQ ID NO: 18, or comprising CDR3 according to (B);
(Ba2) comprising an amino acid sequence that is at least 80% similar to SEQ ID NO: 44;
comprising an amino acid sequence that is at least 80% similar to positions 45 to 49 of SEQ ID NO: 44;
comprising an amino acid sequence that is at least 80% similar to positions 67-73 of SEQ ID NO: 44;
a TCR alpha chain variable region comprising an amino acid sequence at least 80% similar to positions 107-121 of SEQ ID NO: 44, and/or (Bb2) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 46;
comprising an amino acid sequence that is at least 80% similar to positions 44-49 of SEQ ID NO: 46;
comprising an amino acid sequence that is at least 80% similar to positions 67-71 of SEQ ID NO: 46;
A TCR according to any one of items 1 to 5, comprising a TCR beta chain variable region comprising an amino acid sequence at least 80% similar to positions 108-122 of SEQ ID NO: 46.
7. 7. The TCR according to any one of items 1 to 6, further comprising (i) a TCR alpha chain constant region, and/or (ii) a TCR beta chain constant region.
8. The TCR comprising CDR3 according to (A) is
(Aa3) comprising an amino acid sequence that is at least 80% similar to SEQ ID NO: 20;
comprising an amino acid sequence that is at least 80% similar to positions 47-51 of SEQ ID NO: 20;
comprising an amino acid sequence that is at least 80% similar to positions 69-75 of SEQ ID NO: 20;
a TCR alpha chain comprising an amino acid sequence at least 80% similar to positions 109-123 of SEQ ID NO: 20, and/or (Ab3) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 22;
comprising an amino acid sequence that is at least 80% similar to positions 46-50 of SEQ ID NO: 22;
comprising an amino acid sequence that is at least 80% similar to positions 68-73 of SEQ ID NO: 22;
Said TCR comprises a TCR beta chain comprising an amino acid sequence at least 80% similar to positions 110-122 of SEQ ID NO: 22, or comprises CDR3 according to (B),
(Ba3) comprising an amino acid sequence that is at least 80% similar to SEQ ID NO: 48;
comprising an amino acid sequence that is at least 80% similar to positions 45 to 49 of SEQ ID NO: 48;
comprising an amino acid sequence that is at least 80% similar to positions 67-73 of SEQ ID NO: 48;
a TCR alpha chain comprising an amino acid sequence at least 80% similar to positions 107-121 of SEQ ID NO: 48, and/or (Bb3) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 50;
comprising an amino acid sequence that is at least 80% similar to positions 44-49 of SEQ ID NO: 50;
comprising an amino acid sequence that is at least 80% similar to positions 67-71 of SEQ ID NO: 50;
8. A TCR according to any one of items 1 to 7, comprising a TCR beta chain comprising an amino acid sequence at least 80% similar to positions 108-122 of SEQ ID NO: 50.
9. (A) covalently linked to each other to form a TCR heterodimer or multimer;
at least one TCR alpha chain or subregion thereof according to (Aa), (Aa1), (Aa2), or (Aa3) and according to (Ab), (Ab1), (Ab2), or (Ab3) at least one TCR beta chain or subregion thereof, or (B) covalently linked to each other to form a TCR heterodimer or multimer;
at least one TCR alpha chain or subregion thereof according to (Ba), (Ba1), (Ba2), or (Ba3); and at least one TCR alpha chain or subregion thereof according to (Bb), (Bb1), (Bb2), or (Bb3) 9. A TCR according to any one of items 1 to 8, comprising at least one TCR beta chain or subregion thereof.
10. 10. A TCR according to any one of items 1 to 9, wherein said TCR is selected from the group consisting of a native TCR, a TCR variant, a TCR fragment, and a TCR construct.
11. The TCR according to any one of items 1 to 10, which is water-soluble.
12. TCR according to any one of items 1 to 11, further comprising at least one molecular marker.
13. A nucleic acid molecule encoding the TCR according to any one of items 1 to 12.
14. at least 80% identical to any one of the nucleic acid sequences of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21; or SEQ ID NO: 31, 33, 35, 37, 39; 14. The nucleic acid of item 13, comprising a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of any one of 41, 43, 45, 47, or 49.
15. A vector comprising the nucleic acid molecule according to item 13 or 14.
16. A host cell comprising a TCR according to any one of items 1 to 12, a nucleic acid molecule according to item 13 or 14, or a vector according to item 15.
17. Lymphoblastoid cell lines, cytotoxic T lymphocytes (CTL), CD8+ T cells, CD4+ T cells, T memory stem cells ( _TSCM ), natural killer (NK) cells, natural killer T (NKT) cells, and gamma 17. The host cell according to item 16, selected from lymphocytes including, but not limited to, delta-T cells.
18. Obtaining a TCR according to any one of items 1 to 12, comprising incubating a host cell according to item 16 or 17 under conditions that cause expression of said TCR, and purifying said TCR. method for.
19.
(i) a TCR according to any one of items 1 to 12;
(ii) the nucleic acid molecule according to item 13 or 14;
(iii) a vector according to item 15; and/or (iv) one or more of the host cells according to item 16 or 17, and optionally a pharmaceutical excipient. Composition for use.
20. 20. The pharmaceutical composition according to item 19, further comprising a checkpoint inhibitor.
21. 21. The pharmaceutical composition according to item 20, wherein the checkpoint inhibitor is selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.
22. A TCR according to any one of items 1 to 12, a nucleic acid molecule according to items 13 or 14, a vector according to item 15, and/or a host according to items 16 or 17 for use as a medicament. cell.
23. A TCR according to any one of items 1 to 12, a nucleic acid molecule according to items 13 or 14, a vector according to item 15 for use in the detection, diagnosis, prognosis, inhibition, and/or treatment of cancer. , and/or the host cell according to item 16 or 17.
24. Cancers include melanoma, bladder carcinoma, colon carcinoma, breast adenocarcinoma, sarcoma, prostate cancer, uterine cancer, uveal cancer, uveal melanoma, squamous cell head and neck cancer, synovial carcinoma, Ewing's sarcoma, triple negative breast cancer, and thyroid cancer. Cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck Cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct carcinoma, breast cancer, bladder cancer, myeloid leukemia, acute lymphoblastic leukemia, acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma , bone cancer, brain tumor, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile ducts, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear. cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, Pharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, gastrointestinal cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureteral cancer, and The TCR, nucleic acid molecule, vector, or host cell according to item 23, selected from the group consisting of urinary bladder cancer.
25. Prevention and/or treatment of cancer is
(i) a TCR according to any one of items 1 to 12;
(ii) the nucleic acid molecule according to item 13 or 14;
(iii) the vector according to item 15;
(iv) providing one or more of a host cell according to item 16 or 17; and (v) a pharmaceutical composition according to any one of items 19 to 21; and requiring the same. TCR, nucleic acid, vector, and/or host cell for use in item 23 or 24, comprising administering to a subject at least one of (i) to (v).
26. Prevention and/or treatment of cancer is
(1) providing a sample of a subject, the sample comprising lymphocytes;
(2)
(i) a TCR according to any one of items 1 to 12;
(ii) the nucleic acid molecule according to item 13 or 14;
(iii) the vector according to item 15;
(iv) providing one or more of the host cells according to item 16 or 17; and (v) the pharmaceutical composition according to any one of items 19 to 21;
(3) introducing one or more of (i) to (v) of step (2) into the lymphocytes of step (1), thereby obtaining modified lymphocytes; and (4) introducing the modified lymphocytes; A TCR according to any one of items 1 to 12 for use in any one of items 23 to 25, comprising administering the modified lymphocytes of step (3) to a subject or patient in need thereof; The nucleic acid molecule according to item 13 or 14, the vector according to item 15, and/or the host cell according to item 16 or 17.
27. providing a sample of interest, said sample comprising one or more cells;
The sample;
(i) a TCR according to any one of items 1 to 12;
(ii) contacting a host cell according to item 16 or 17, and/or (iii) a pharmaceutical composition according to any one of items 19 to 21, thereby forming a complex; and 1. A method of detecting the presence of cancer in a subject in vitro, the detection of the complex being indicative of the presence of cancer in the subject.
28. Use of a TCR according to any one of items 1 to 12, a nucleic acid molecule according to items 13 or 14, and/or a vector according to item 15 for producing modified lymphocytes.

The embodiments characterizing the invention are described herein, shown in the drawings, illustrated in the examples, and reflected in the claims.

It is noted that as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. There must be. Thus, for example, reference to "a reagent" includes one or more of a variety of such reagents, and reference to "the method" herein includes one or more of a variety of such reagents. Contains references to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the methods described.

Unless indicated otherwise, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

The term "and/or" wherever used herein includes the meanings of "and," "or," and "all or any other combination of the elements connected by the term." .

The term "about" or "approximately" as used herein means within 20%, preferably within 10%, more preferably within 5% or 2% of a given value or range.

Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are used in the description It will be understood that the inclusion of an integer or step, or group of integers or steps, does not imply the exclusion of any other integer or step, or group of integers or steps. As used herein, the term "comprising" refers to the term "containing" or "including," or as used herein sometimes "having." (having)”.

As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not significantly affect the essential novel features of the claim.

In each case herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms.

It is to be understood that this invention is not limited to the particular methodologies, protocols, reagents, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. .

All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.) cited throughout the text of this specification, whether cited above or below, are are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an understanding that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference is inconsistent or inconsistent with the present specification, the present specification will supersede any such material.

Lymphoblastoid cell lines (LCLs; EBV-transformed B cells) expressing the HLA-A ^* 24:02 encoding molecule were electroporated with either PRAME-encoding ivtRNA or water, and the PRAME _301-309 peptide or Either unrelated peptides were loaded. These cells were used as targets in co-culture assays with TCR T402-93 or TCR T116-49 transgenic T cells. Non-transduced T cells served as a negative control. After 24 hours (h) of incubation, IFN-γ release by TCR transgenic T cells was assessed by standard ELISA. This experiment was performed using two different donors. One representative experiment is shown. CD8 ⁺ T cells expressing either TCR T402-93 or TCR T116-49 were incubated with HLA-A ^* 24-positive PRAME-positive tumor cell lines (K562, Mel624.38, CMK, SKHEP1) or HLA-A ^* 24-positive Co-cultured with any of the PRAME-negative tumor cell lines (Colo678, MCF-7, 22RV1). Non-transduced CD8 ⁺ T cells served as a negative control. HLA-A ^* 24-positive LCL electroporated with either PRAME-encoding ivtRNA or water and carrying either the PRAME _301-309 peptide or an unrelated peptide were included as internal target controls. Activation of transgenic TCR-expressing T cells was assessed by standard ELISA measuring IFN-γ release in [pg/ml] after 24 hours of co-culture. Duplicate mean values are shown with standard deviations. This experiment was performed using two different donors. One representative experiment is shown. Values above 4000 pg were extrapolated using a cubic polynomial. Red labeled tumor cell lines were incubated with either TCR T402-93 or TCR T116-49 transgenic T cells and with non-transduced T cells. Cells were monitored using a live cell imaging system over a period of 105 hours to assess killing of red labeled tumor cells mediated by TCR transgenic T cells. Total integrated intensity (RCU (red calibration units) x μm ² /image) was calculated using IncuCyte ZOOM® software. Each measurement point represents the average of three technical replicates. This experiment was performed using two different donors. One representative experiment is shown. Red labeled tumor cell lines were incubated with either TCR T402-93 or TCR T116-49 transgenic T cells and with non-transduced T cells. Cells were monitored using a live cell imaging system over a period of 105 hours to assess killing of red labeled tumor cells mediated by TCR transgenic T cells. Total integrated intensity (RCU (red calibration units) x μm ² /image) was calculated using IncuCyte ZOOM® software. Each measurement point represents the average of three technical replicates. This experiment was performed using two different donors. One representative experiment is shown. HLA-A ^* 24-positive LCLs were loaded with a titer (10 ⁻⁵ M to 10 ⁻⁹ M) of PRAME _301-309 peptide and co-incubated with either TCR T402-93 or TCR T116-49 expressing CD8 ⁺ T cells. Cultured. A standard ELISA was performed 24 hours later to assess IFN-γ release by T cells. Maximum IFN-γ release per effector cell sample was set at 100%. Based on this, relative IFN-γ release was calculated. This experiment was performed using two different donors. One representative experiment is shown. A threonine scanning assay was performed on the 9-mer PRAME _301-309 (LYVDSLFFL) peptide recognized by TCR T402-93 and TCR T116-49. The amino acids contained in the PRAME _301-309 peptide were successively replaced by threonine (replaced aa are shown in bold). HLA-A ^* 24-positive LCLs loaded with modified peptide (10 ⁻⁵ M) and co-cultured with T cells expressing either TCR T402-93 or TCR T116-49 and with non-transduced T cells. IFN-γ secretion was assessed by standard ELISA after 24 hours of incubation. Duplicate mean values are shown with standard deviations. This experiment was performed using two different donors. One representative experiment is shown. Peptides with up to 3 amino acid differences compared to the wild type 9-mer PRAME _301-309 (LYVDSLFFL) peptide were selected using the Expitope 2.0® tool. Mismatched peptides were loaded on HLA-A ^* 24 positive LCL (10 ⁻⁵ M) and tested for recognition by either TCR T402-93 or TCR T116-49 transgenic T cells. PRAME _301-309- bearing LCL was included as an internal positive control. T cell activation was assessed using a standard ELISA IFN-γ after 24 hours of incubation. This experiment was performed using two different donors. One representative experiment is shown. T cells transduced with TCR T116-49 were treated with a cell library of 52 LCLs covering the most frequent HLA-A, -B, and -C alleles in German and US/European Caucasian populations. co-cultured. In addition, the same 52 LCLs were loaded with PRAME _301-309 peptide and tested in co-culture with TCR T116-49 transduced T cells. A standard ELISA was performed after 24 hours of incubation to assess IFN-γ release by T cells. This experiment was performed using two different donors. One representative experiment is shown.

The invention is further illustrated by the following examples. However, the Examples and the specific embodiments described therein are not to be construed as limiting the invention to such specific embodiments.

[Example 1]
Isolation of PRAME _301-309- specific HLA-A24-restricted TCRs An in vitro priming procedure was used to isolate any desired HLA-restricted and antigen-specific T cell clones. The priming system used HLA-A ^* 24:02 negative healthy donor mature dendritic cells (mDCs) as antigen-presenting cells and autologous CD8-enriched T cells as responder cells. In vitro transcribed RNA (ivtRNA) encoding the full-length human PRAME amino acid sequence served as a source of specific antigen. At the same time, human HLA-A ^* 24:02-encoded ivtRNA (sequence derived from https://www.ebi.ac.uk/ipd/imgt/hla/) was used as a source of restrictive elements to infect mDCs. transfected to assemble homologous priming for this dedicated HLA allele (as described in WO 2007/017201). After electroporation into mDCs, PRAME-encoded ivtRNAs were transcribed into full-length proteins, which were subsequently processed and presented as peptides by transgenic HLA-A ^* 24 molecules expressed by transfected mDCs. In vitro co-culture of T cells with ivtRNA-transfected mDCs from the same donor led to de novo induction of antigen-specific T cells that served as a source of the corresponding TCR.

Allogeneic T cell priming techniques using HLA-A ^* 24:02-encoded ivtRNA-transfected and PRAME ivtRNA-transfected mDCs with allogeneic HLA-A ^* 24:02-encoded molecules according to the following protocol. This was accomplished using peptide presentation.

HLA-A ^* 24:02/PRAME Priming Monocytes were derived from HLA-A ^* 24:02-negative healthy donors and the corresponding mDCs were purified using the protocol of Jonuleit et al., Eur. J. Immunol. 1997 , 27:3135-3142) using appropriate maturation cocktails. mDCs were co-electroporated with 20 μg ivtRNA encoding PRAME and 20 μg ivtRNA encoding HLA-A ^* 24 molecules. The prepared mDCs were then co-cultured with autologous CD8 ⁺ T cells at a 1:10 ratio for approximately 14 days in appropriate cell culture medium supplemented with IL-2 (50 units/ml). PRAME _301-309 specific T cells were then identified using HLA-A ^* 24:02 PRAME _301-309 multimers and isolated by single cell sorting using FACS technology. After identification of a promising T cell clone recognizing the desired PRAME _301-309 epitope on the HLA-A ^* 24 molecule, the corresponding T cell receptor (TCR) sequence was analyzed by next generation sequencing (NGS). The identified HLA-A ^* 24-restricted PRAME _301-309- specific TCRs (T402-93 and T116-49) were expressed in recipient T cells and characterized in terms of function and specificity.

[Example 2]
Evaluation of antigen specificity LCL expressing HLA-A ^* 24:02 encoding molecules were loaded with specific PRAME _301-309 peptides or unrelated peptides at a concentration of 10 ⁻⁵ M. Additionally, the same HLA-A ^* 24 positive LCLs were electroporated with either PRAME-encoding ivtRNA or water as a negative control. Each target cell line was incubated with either TCR T402-93 or TCR T116-49 at a 1:2 effector:target (E:T) ratio using 10,000 T cells and 20,000 targets per 96 well. were co-cultured with transduced T cells. Untransduced T cells (UT) were included as a negative control. After 24 hours of co-culture, IFN-γ released by T cells was measured by standard ELISA.

result:
Both TCR T402-93 and TCR T116-49 transduced T cells recognized the specific PRAME _301-309 peptide as well as PRAME transfected LCL. Of note, T cells expressing TCR T116-49 exhibited higher levels of IFN-γ release following incubation with positive targets compared to TCR T402-93 transgenic T cells. No recognition of LCL loaded with unrelated peptides and electroporated with water was observed (Figure 1).

[Example 3]
Evaluation of IFN-γ release by tumor cell recognition T cells Effector T cells transduced with either TCR T402-93 or TCR T116-49 were incubated with PRAME positive tumor cell lines (K562, Mel624.38, CMK, SKHEP1). or co-cultured with any of the PRAME-negative tumor cell lines (Colo678, MCF-7, 22RV1). Among the selected tumor cell lines, CMK and SKHEP1 cell lines are endogenously positive for HLA-A ^* 24, while the other five cell lines are endogenously positive for HLA-A ^* 24. It is negative. Therefore, these five cell lines were transduced with HLA-A ^* 24 (K562, Mel624.38, 22RV1) or transfected with ivtRNA encoding HLA-A ^* 24 molecules (Colo678 and MCF- Tested after any of 7). Non-transduced CD8 ⁺ T cells served as a negative control. HLA-A ^* 24-positive LCL electroporated with either PRAME-encoding ivtRNA or water and carrying either the PRAME _301-309 peptide or an unrelated peptide were included as internal controls. T cells and target cells were co-cultured at a 1:1 E:T ratio (10,000 E/10,000 T/96 wells). Activation of transgenic TCR-expressing T cells was assessed by standard ELISA measuring IFN-γ release in [pg/ml] after 24 hours of co-culture. Values above 4000 pg were extrapolated using a cubic polynomial.

result:
T cells transduced with TCR T116-49 showed recognition of all PRAME-positive tumor cells tested, whereas T cells transduced with TCR T402-93 showed recognition of all PRAME-positive tumor cells tested. released high levels of IFN-γ only after co-culture with two species (K562 and Mel624.38). No recognition of any PRAME negative cells was observed in co-culture with T cells transduced with TCR T116-49. In contrast, slight recognition of the PRAME negative cell line (MCF-7) was observed for T cells expressing TCR 402-93 (Figure 2).

Evaluation of killing mediated by T cells To assess killing mediated by TCR transgenic T cells, two PRAME positive tumor cell lines (Mel624.38, SKHEP1) and a PRAME negative tumor cell line (Colo678) were selected as target cells. Among the selected tumor cell lines, the SKHEP1 cell line is intrinsically positive for HLA-A ^* 24, while the other three cell lines are intrinsically HLA-A ^* 24 negative. be. Therefore, SKHEP1 cells were transduced with mCherry (red fluorescent protein) only, while the other two cell lines were transduced with HLA-A ^* 24 linked to mCherry. Red-labeled tumor cells were seeded in 96-well flat bottom plates 2 days before the start of co-culture (Mel624.38 and SKHEP1 5000 cells/well, while Colo678 10000 cells/well). As an internal positive control, the same tumor cell line was additionally loaded with PRAME _301-309 peptide. After adding 10,000 T cells expressing either TCR T402-93 or TCR T116-49 per well, the co-culture plate was transferred to a live cell imaging system (IncuCyte ZOOM® device). Cells were monitored over a total period of 105 hours to assess killing of red labeled tumor cells mediated by TCR transgenic T cells. The total sum of red fluorescence intensities of the subjects in the images, called the total integrated intensity (RCU (red calibration units) x μm ² /image), was calculated using IncuCyte ZOOM® software.

result:
Both TCR-transduced samples affected the growth of the Mel624.38 PRAME-positive cell line, and in contrast, only TCR T116-49-transduced T cells affected the efficient growth of the SKHEP1 PRAME-positive cell line. caused a great deal of murder and injury. Both TCR-transduced samples had no effect on the expansion of PRAME-negative tumor cells. Each target cell line after peptide loading was efficiently killed by TCR-transduced T cells. In contrast, growing target cells were observed for all tumor cell lines when non-transduced T cells were used as effectors in co-cultures (Figure 3).

[Example 4]
Functional Avidity The purpose of the experiment was to measure the functional avidity of the PRAME _301-309 specific TCR. Functional avidity refers to the cumulative strength of multiple affinities of individual non-covalent interactions, such as between a transgenic TCR and a pMHC complex. The functional avidity ^of TCR transgenic T ^cell populations was _determined by half ^- maximal relative Measured as IFN-γ release. T cells and target cells were co-cultured at a 1:1 E:T ratio (10,000 E/10,000 T/96 wells). Non-transduced CD8 ⁺ T cells were used as an internal control to subtract reactivity mediated by the T cell's endogenous TCR and not related to transgenic TCR-specific recognition. A standard ELISA was performed 24 hours later to assess IFN-γ release by T cells. Maximum IFN-γ release per effector cell sample was set at 100%. Based on this, relative IFN-γ release was calculated. This experiment was performed using two different donors. One representative experiment is shown.

result:
T cells transduced with TCR T116-49 showed higher functional avidity and higher sensitivity to the target peptide compared to T cells transduced with TCR T402-93 (Figure 4) .

[Example 5]
TCR recognition motif (threonine scan assay)
The purpose of the experiment was to assess critical residues within the PRAME _301-309 epitope that are essential for direct recognition by the TCR or for peptide binding to HLA-A ^* 24:02-encoded molecules. Amino acid substitution scanning was used to define critical amino acids in the epitope sequence that abolished recognition by the TCR whenever these residues were exchanged with the amino acid threonine. These "fixed" amino acids can be used to define unique TCR recognition motifs. Threonine scanning assays were performed on the 9-mer PRAME _301-309 peptide recognized by TCR T402-93 and TCR T116-49. The amino acids contained in the PRAME _301-309 peptide were successively replaced by threonine. HLA-A ^* 24-positive LCLs were loaded with modified peptide (10 ⁻⁵ M) and wild-type PRAME _301-309 peptide, and co-cultured with T cells expressing either TCR T402-93 or TCR T116-49. It was used in Non-transduced T cells served as internal control. T cells and target cells were co-cultured at a 1:1 E:T ratio (10,000 E/10,000 T/96 wells). To assess IFN-γ secretion by T cells, a standard ELISA was performed after 24 hours of co-culture.

result:
T cells transduced with TCR T116-49 exhibited different TCR recognition motifs with less fixed locations compared to T cells transduced with TCR T402-93 (Figure 5).

[Example 6]
Recognition of mismatched peptides Wild-type 9-mer PRAME _301-309 was determined by in silico analysis using the Expitope 2.0 tool (Expitope 2.0; Jaravine et al. BMC Cancer 2017). We selected 52 peptides containing up to 3 mismatches compared to the epitope. The mismatched peptide was loaded on HLA-A ^* 24-positive LCL (10 ⁻⁵ M) and its recognition by TCR T402-93 and TCR T116-49 transgenic T cells was tested. Wild-type PRAME _301-309 peptide-loaded LCL as well as unloaded LCL were included as internal controls. T cells and target cells were co-cultured at a 1:1 E:T ratio (10,000 E/10,000 T/96 wells). T cell activation was assessed using a standard ELISA IFN-γ after 24 hours of incubation.

result:
The TCR transgenic T cell samples recognized the wild type PRAME _301-309 peptide but not the uncarried target, thus demonstrating the functionality of the transgenic T cells. T cells transduced with TCR T402-93 were also activated by target cells carrying peptides #4, #33, #38, and #42, while T cells transduced with TCR T116-49 releases IFN-γ upon stimulation by LCL carrying mismatched peptide #18 (Figure 6 and Table 2).

[Example 7]
LCL Library A cell library consisting of 52 LCLs covering the most frequent HLA-A, -B, and -C alleles in German and US/European Caucasian populations was established. HLA allele frequencies greater than 0.5% in any of these populations are covered by at least one cell line, and HLA alleles exhibiting a frequency greater than 5% are covered by at least two LCLs (HLA- A ^* Excluding 11:01). The first objective of the experiment was to investigate potential target antigen-independent cross-recognition of frequent HLA allotypes by TCR T116-49. HLA-allocross-recognition can be defined as the ability of a TCR to interact with cognate HLA molecules, whereby these interactions are also described as exhibiting exquisite peptide and HLA specificity. Therefore, 52 LCLs were incubated with T cells expressing TCR T116-49. An additional objective of the experiment was to determine common HLA-A suballeles other than HLA-A ^* 24:02 that can present the PRAME _301-309 epitope and can be recognized by TCR T116-49 transgenic T cells. (HLA-restricted microtyping). Therefore, 52 LCLs were loaded with PRAME _301-309 peptide (10 ⁻⁵ M) and subsequently used as targets in co-culture with TCR T116-49 transgenic T cells. After 24 hours of incubation, a standard ELISA was performed to measure IFN-γ release by T cells.

result:
IFN-γ release by TCR T116-49 transduced T cells was observed after co-culture with HLA-A ^* 24:02 positive LCLs carrying all PRAME _301-309 peptides included in the library. TCR T116-49 transgenic T cells weakly recognized LCL expressing HLA-A ^* 02:02 without peptide loading as well as after PRAME _301-309 peptide loading, indicating that the HLA-A ^* 02:02 allele suggested potential HLA-allocross recognition for. Two types of LCL expressing HLA-A ^* 02:17 were recognized by TCR T116-49 transgenic T cells only after PRAME _301-309 peptide loading, indicating that the PRAME _301-309 epitope was HLA-A ^* 02: 17 encoding molecules, leading to activation of T116-49 transgenic T cells.

Sequences (in case of discrepancies, the following sequences govern those in the sequence listing according to the WIPO ST.25 standard):

Claims (15)

in a polypeptide comprising an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), or in a part of said polypeptide, or in each HLA- A T cell receptor (TCR) capable of binding to the A-linked form,
(A)
(Aa) CDR3 of the TCR alpha chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 12, and/or (Ab) CDR3 of the TCR beta chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 14; or (B)
(Ba) CDR3 of the TCR alpha chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 40, and/or (Bb) CDR3 of the TCR beta chain comprising an amino acid sequence at least 80% similar to SEQ ID NO: 42.
Including TCR. The TCR comprising CDR3 according to (A) is
(Aa1) CDR1 of a TCR alpha chain comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 4, and/or a TCR alpha comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 8; CDR2 of the TCR beta chain, and/or (Ab1) at least 80% similar to the amino acid sequence of SEQ ID NO: 10; CDR2 of the TCR beta chain containing the amino acid sequence
or comprising a CDR3 according to (B),
(Ba1) CDR1 of a TCR alpha chain comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 32, and/or a TCR alpha comprising an amino acid sequence at least 80% similar to the amino acid sequence of SEQ ID NO: 36; CDR2 of the TCR beta chain, and/or (Bb1) at least 80% similar to the amino acid sequence of SEQ ID NO: 38; CDR2 of the TCR beta chain containing the amino acid sequence
The TCR of claim 1, further comprising: The TCR according to claim 1 or 2, wherein HLA-A is an HLA-A ^* 24 or HLA-A ^* 02 encoding molecule. 4. The method according to any one of claims 1 to 3, wherein binding of the TCR to the polypeptide or a portion thereof, or an HLA-A bound form thereof, induces IFN-gamma secretion by cells containing the TCR. T.C.R. Said induction of IFN-gamma secretion of cells comprising said TCR is directed to or into a polypeptide comprising an amino acid sequence according to the amino acid sequence LYVDSLFFL (SEQ ID NO: 2), in which not more than 4 amino acids have been substituted. 5. A TCR according to claim 4, wherein the binding to the portion or to the respective HLA-A bound form of said polypeptide or portion thereof is at least 5 times higher compared to control cells without said TCR. . The TCR comprising CDR3 according to (A) is
(Aa2) comprises an amino acid sequence that is at least 80% similar to SEQ ID NO: 16;
comprising an amino acid sequence that is at least 80% similar to positions 47-51 of SEQ ID NO: 16;
comprising an amino acid sequence that is at least 80% similar to positions 69-75 of SEQ ID NO: 16;
a TCR alpha chain variable region comprising an amino acid sequence at least 80% similar to positions 109-123 of SEQ ID NO: 16, and/or (Ab2) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 18;
comprising an amino acid sequence that is at least 80% similar to positions 46 to 50 of SEQ ID NO: 18;
comprising an amino acid sequence that is at least 80% similar to positions 68-73 of SEQ ID NO: 18;
said TCR comprising a TCR beta chain variable region comprising an amino acid sequence at least 80% similar to positions 110-122 of SEQ ID NO: 18, or comprising CDR3 according to (B);
(Ba2) comprising an amino acid sequence that is at least 80% similar to SEQ ID NO: 44;
comprising an amino acid sequence that is at least 80% similar to positions 45 to 49 of SEQ ID NO: 44;
comprising an amino acid sequence that is at least 80% similar to positions 67-73 of SEQ ID NO: 44;
a TCR alpha chain variable region comprising an amino acid sequence at least 80% similar to positions 107-121 of SEQ ID NO: 44, and/or (Bb2) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 46;
comprising an amino acid sequence that is at least 80% similar to positions 44-49 of SEQ ID NO: 46;
comprising an amino acid sequence that is at least 80% similar to positions 67-71 of SEQ ID NO: 46;
6. The TCR of any one of claims 1 to 5, comprising a TCR beta chain variable region comprising an amino acid sequence at least 80% similar to positions 108-122 of SEQ ID NO: 46. The TCR comprising CDR3 according to (A) is
(Aa3) comprising an amino acid sequence that is at least 80% similar to SEQ ID NO: 20;
comprising an amino acid sequence that is at least 80% similar to positions 47-51 of SEQ ID NO: 20;
comprising an amino acid sequence that is at least 80% similar to positions 69-75 of SEQ ID NO: 20;
a TCR alpha chain comprising an amino acid sequence at least 80% similar to positions 109-123 of SEQ ID NO: 20, and/or (Ab3) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 22;
comprising an amino acid sequence that is at least 80% similar to positions 46-50 of SEQ ID NO: 22;
comprising an amino acid sequence that is at least 80% similar to positions 68-73 of SEQ ID NO: 22;
Said TCR comprises a TCR beta chain comprising an amino acid sequence at least 80% similar to positions 110-122 of SEQ ID NO: 22, or comprises CDR3 according to (B),
(Ba3) comprising an amino acid sequence that is at least 80% similar to SEQ ID NO: 48;
comprising an amino acid sequence that is at least 80% similar to positions 45 to 49 of SEQ ID NO: 48;
comprising an amino acid sequence that is at least 80% similar to positions 67-73 of SEQ ID NO: 48;
a TCR alpha chain comprising an amino acid sequence at least 80% similar to positions 107-121 of SEQ ID NO: 48, and/or (Bb3) comprising an amino acid sequence at least 80% similar to SEQ ID NO: 50;
comprising an amino acid sequence that is at least 80% similar to positions 44-49 of SEQ ID NO: 50;
comprising an amino acid sequence that is at least 80% similar to positions 67-71 of SEQ ID NO: 50;
7. A TCR according to any one of claims 1 to 6, comprising a TCR beta chain comprising an amino acid sequence at least 80% similar to positions 108-122 of SEQ ID NO:50. (A) covalently linked to each other to form a TCR heterodimer or multimer;
at least one TCR alpha chain or subregion thereof according to (Aa), (Aa1), (Aa2), or (Aa3) and according to (Ab), (Ab1), (Ab2), or (Ab3) at least one TCR beta chain or subregion thereof, or (B) covalently linked to each other to form a TCR heterodimer or multimer;
at least one TCR alpha chain or subregion thereof according to (Ba), (Ba1), (Ba2), or (Ba3); and at least one TCR alpha chain or subregion thereof according to (Bb), (Bb1), (Bb2), or (Bb3) 8. A TCR according to any one of claims 1 to 7, comprising at least one TCR beta chain or subregion thereof. A nucleic acid molecule encoding a TCR according to any one of claims 1 to 8. at least 80% identical to any one of the nucleic acid sequences of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21; or SEQ ID NO: 31, 33, 35, 37, 39; 10. The nucleic acid of claim 9, comprising a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of any one of 41, 43, 45, 47, or 49. A vector comprising the nucleic acid molecule according to claim 9 or 10. A host cell comprising a TCR according to any one of claims 1 to 8, a nucleic acid molecule according to claims 9 or 10, or a vector according to claim 11. (i) TCR according to any one of claims 1 to 8;
(ii) the nucleic acid molecule according to claim 9 or 10;
(iii) a vector according to claim 11; and/or (iv) one or more of the host cells according to claim 12, and optionally a pharmaceutical excipient. Composition for use. A TCR according to any one of claims 1 to 8, a nucleic acid molecule according to claims 9 or 10, a vector according to claim 11 and/or according to claim 12 for use as a medicament. host cells. A TCR according to any one of claims 1 to 8, a nucleic acid molecule according to claim 9 or 10, for use in the detection, diagnosis, prognosis, prevention and/or treatment of cancer, according to claim 11. A vector according to claim 12 and/or a host cell according to claim 12.